Systems and methods for nucleic acid sequence assembly

ABSTRACT

Methods, processes, and particularly computer implemented processes and computer program products are provided for use in the analysis of genetic sequence data. The processes and products are employed in the assembly of shorter nucleic acid sequence data into longer linked and preferably contiguous genetic constructs, including large contigs, chromosomes and whole genomes.

CROSS REFERENCE TO RELATED APPLICATION

This application is continuation of U.S. patent application Ser. No.15/801,868, entitled “SYSTEMS AND METHODS FOR NUCLEIC ACID SEQUENCEASSEMBLY,” filed Nov. 2, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/752,773, entitled “PROCESSES AND SYSTEMS FORNUCLEIC ACID SEQUENCE ASSEMBLY,” filed Jun. 26, 2015, which claimspriority to U.S. Provisional Application No. 62/017,589, filed Jun. 26,2014, both of which are hereby incorporated by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing text copy submitted herewith via EFS-Web wascreated on Dec. 30, 2021, is entitled 1043715013US02_ST25.txt, is 1kilobyte in size and is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

One of the significant challenges associated with high throughput nextgeneration sequencing technologies is in assembling relatively shortsequence reads into much longer contiguous sequences. Genomic sequenceassembly processes are often analogized to having a novel that is cutinto small pieces, which one must then be reassembled into the completenovel. In sequence assembly, this is generally accomplished by piecingtogether overlapping sub-segments of the whole sequence that is to beassembled. As will be appreciated, that assembly process becomes easierwhen the pieces are larger and less ambiguous. For example, it would beeasier to assemble a novel based upon full paragraphs, pages or chaptersthan it would from individual sentences, sentence fragments or randomwords or parts of words. Likewise, with nucleic acid sequencing, theshorter the individual sequence read, the more difficult it becomes toassemble multiple reads into a longer contiguous sequence.

While next generation sequencing technologies are capable of generatingvast amounts of sequence data, e.g., generating 1 terabase of sequencedata within a single run, they are plagued by the difficulties ofproducing only short sequence read lengths. In particular, thesesequencing technologies generally acquire sequence data in reads of 100contiguous bases or less (or when sequenced as paired end sequencing, upto approximately 200 bases). These reads then must be assembled intolonger contiguous sequences. While certain sequencing technologiesprovide longer sequence reads of 800, 1000, 5000, or even 20,000 basesin length, this often comes at the price of system throughput, yieldingonly hundreds of megabases of sequence data per run.

A number of processes and algorithms have been employed in assemblingrelatively short sequence reads into longer contiguous sequences. Ingeneral, these short sequence reads typically overlap with a number ofother short sequence reads to provide redundant coverage over differentparts of the overall longer sequence (referred to as “coverage” or “foldcoverage”). At a high level, the overlapping sequences from numerousreads are used to piece together the longer sequence information. Inmany cases, existing known reference sequences, e.g., from the samespecies, are available. These reference sequences may be used as ascaffold for mapping the shorter sequence reads onto a larger genome orgenome fragment. The multifold sequence coverage is then layered on topof the scaffold to provide a relatively high confidence sequence.

In many cases, however, reference sequences may not be available,requiring de novo assembly of the genome or genomic fragment beingsequenced. Moreover, in many cases, such reference sequences are nothelpful because the target of the sequencing efforts it to detectmutations in the target sequence. For instance, in the case where thetarget sequence is from a cancer, it is import to ascertain whichmutations, insertions, and deletions are in the target sequence as a wayof diagnosing cancer stage or some other attribute of the cancer. Assuch, making use of references sequences is not always useful insequencing efforts.

Accordingly, described herein are improved methods, processes andsystems for the assembly of sequence reads into larger genome or genomefragment sequences, and particularly, for de novo assembly of sequenceinformation into larger sequence contexts, including for example, wholechromosome or even whole genome contexts.

SUMMARY

Described herein are improved genetic sequence assembly processes usedfor assembly of sequence reads from a variety of different sequencingsystems, including e.g., both short and long read sequencing systems.The processes described herein may be used in both de novo assemblyprocesses and in re-sequencing assembly processes or assembly processesagainst known reference sequences.

The methods, processes and systems described herein employ sequencereads with associated barcode sequences to assist in the overallassembly process at one or more different assembly steps.

In certain aspects, provided are methods of assembling nucleic acidsequence reads into larger contiguous sequences. The methods compriseidentifying a first subset of sequence reads that comprise bothoverlapping sequences and a common barcode sequence in a computerimplemented system comprising a first data structure comprising aplurality of sequence reads derived from a larger contiguous nucleicacid. Sequence reads derived from a common fragment of the largercontiguous nucleic acid comprise a common barcode sequence. The firstsubset of sequence reads is aligned to provide a contiguous linearnucleic acid sequence

Also provided are methods of assembling nucleic acid sequence reads intolarger contiguous sequences. Such methods identify a first subsequencefrom a set of overlapping sequence reads in a computer implementedsystem comprising a first data structure comprising a plurality ofsequence reads derived from a larger contiguous nucleic acid. The firstsubsequence is then extended to one or more adjacent or overlappingsequences based upon the presence of a barcode sequence on the adjacentsequence that is common to the first subsequence. A linear nucleic acidsequence is then provided that comprises the first subsequence and theone or more adjacent sequences.

One aspect of the present disclosure provides a sequencing methodcomprising, at a computer system having one or more processors, andmemory storing one or more programs for execution by the one or moreprocessors, implementing a method in which a plurality of sequence readsis obtained. The plurality of sequence reads comprises a plurality ofsets of sequence reads. Each respective sequence read in a set ofsequence reads includes (i) a first portion that corresponds to a subsetof a larger contiguous nucleic acid and (ii) a common second portionthat forms an identifier that is independent of the sequence of thelarger contiguous nucleic acid and that identifies a partition, in aplurality of partitions, in which the respective sequence read wasformed. Each respective set of sequence reads in the plurality of setsof sequence reads is formed in a partition in the plurality ofpartitions and each partition includes one or more fragments (e.g., twoor more, three or more, ten or more) of the larger contiguous nucleicacid that is used as the template for each respective sequence read inthe partition.

In the method, a respective set of k-mers is created for each sequenceread in the plurality of sequence reads. The sets of k-mers collectivelycomprise a plurality of k-mers. The identifier of the sequence read foreach k-mer in the plurality of k-mers is retained. In many instances,there are multiple such sequencing read identifiers for at least some ofthe k-mers. The value k is less than the average length of the sequencereads in the plurality of sequence reads. Each respective set of k-mersincludes some (e.g., at least eighty percent) of the possible k-mers oflength k of the first portion of the corresponding sequence read.

In the method, there is tracked, for each respective k-mer in theplurality of k-mers, an identity of each sequence read in the pluralityof sequence reads that contains the respective k-mer and the identifierof the set of sequence reads that contains the sequence read.

In the method, all or a portion (e.g., at least 1 percent, at least 5percent, at least fifty percent) of the plurality k-mers are graphed asa graph comprising a plurality of nodes connected by a plurality ofdirected arcs. Each node comprises an uninterrupted set of k-mers in theplurality of k-mers of length k with k−1 overlap. Each arc connects anorigin node to a destination node in the plurality of nodes. A finalk-mer of an origin node has k−1 overlap with an initial k-mer of adestination node. A first origin node has a first directed arc with botha first destination node and a second destination node in the pluralityof nodes.

In the method, a determination is made as to whether to merge the originnode with the first destination node or the second destination node inorder to derive a contig sequence that is more likely to berepresentative of a portion of the larger contiguous nucleic acid. Thecontig sequence comprises (i) the origin node and (ii) one of the firstdestination node and the second destination node. The determining usesat least the identifiers of the sequence reads for k-mers in the firstorigin node, the first destination node, and the second destinationnode.

Another aspect provides a computing system, comprising one or moreprocessors, memory storing one or more programs to be executed by theone or more processors, the one or more programs comprising instructionsfor executing the method described above.

Another aspect of the present disclosure provides a sequencing methodcomprising, at a computer system having one or more processors, andmemory storing one or more programs for execution by the one or moreprocessors, obtaining a plurality of sequence reads. The plurality ofsequence reads comprises a plurality of sets of sequence reads. Eachrespective sequence read in a set of sequence reads includes (i) aunique first portion that corresponds to a subset of a larger contiguousnucleic acid and (ii) a common second portion that forms an identifierthat is independent of the sequence of the larger contiguous nucleicacid and that identifies a partition, in a plurality of partitions, inwhich the respective sequence read was formed. Each respective set ofsequence reads in the plurality of sets of sequence reads is formed in apartition in the plurality of partitions. Each such partition includesone or more fragments of the larger contiguous nucleic acid that is usedas the template for each respective sequence read in the partition.

In the method, a respective set of k-mers is created for each sequenceread in the plurality of sequence reads. The sets of k-mers collectivelycomprise a plurality of k-mers. The identifier of the sequence read foreach k-mer in the plurality of k-mers is retained. The value k is lessthan the average length of the sequence reads in the plurality ofsequence reads. Each respective set of k-mers includes at least some of(e.g., at least eighty percent of) the possible k-mers of the firstportion of the corresponding sequence read.

In the method, there is tracked, for each respective k-mer in theplurality of k-mers, an identity of the each sequence read in theplurality of sequence reads that contains the respective k-mer. A firstpath is identified. The first path comprises a first set of k-mers withk−1 overlap in the plurality of k-mers. A second path is identified. Thesecond path comprises a second set of k-mers with k−1 overlap in theplurality of k-mers. The first path intersects the second path (e.g., asillustrated in FIG. 15), thereby forming the set of branch segmentscomprising: a left portion of the first path, a right portion of thefirst path, a left portion of the second path and a right portion of thesecond path. The identifiers associated with the k-mers of the set ofbranch segments are used to verify the connectivity of the branchsegments. In some embodiments, this comprises evaluating a number ofidentifiers shared between each possible branch segment pair in the setof branch segments.

In some embodiments, a partition in the plurality of partitionscomprises at least 1000 molecules with the common second portion, andeach molecule in the at least 1000 molecules includes a primer sequencecomplementary to at least a portion of the larger contiguous nucleicacid.

In some embodiments, the identifier in the second portion of eachrespective sequence read in the set of sequence reads encodes a commonvalue selected from the set {1, . . . , 1024}, the set {1, . . . ,4096}, the set {1, . . . , 16384}, the set {1, . . . , 65536}, the set{1, . . . , 262144}, the set {1, . . . , 1048576}, the set {1, . . . ,4194304}, the set {1, . . . , 16777216}, the set {1, . . . , 67108864},or the set {1, . . . , 1×10¹²}.

In some embodiments, the identifier is an N-mer, where N is an integerselected from the set {4, . . . , 20}.

In some embodiments, an average sequence read length of the plurality ofsequence reads is between 40 bases and 200 bases or between 60 bases and140 bases.

In some embodiments, the plurality of sequence reads collectivelyprovide at least 15× coverage for the larger contiguous nucleic acid,more than ten percent of the k-mers in the plurality of k-mers are frommore than one source sequence read in the plurality sequence reads, andthe identifier of each such source sequence read for each k-merrepresented by more than one source sequence read is retained.

In some embodiments, the plurality of sequence reads collectivelyprovide at least 25×coverage for the larger contiguous nucleic acid,more than thirty percent of the plurality of k-mers are from more thanone source sequence read in the plurality sequence reads, and theidentifier of each such source sequence read for each k-mer representedby more than one source sequence read is retained.

In some embodiments, the sequence reads in the plurality of sequencereads encode between 75 and 125 bases of the larger contiguous nucleicacid and k is an odd integer between 5 and 73.

In some embodiments, a set of sequence reads in the plurality ofsequence reads comprises more than 100 sequence reads, each with thesame common second portion.

In some embodiments, the larger contiguous nucleic acid is a chromosomeand/or is greater than 40 million base pairs in length.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a schematic illustration of alignment of overlappingsequence reads.

FIG. 1B shows a sample de Bruijn initial assembly graph based upon thesequence reads, with primary path identification.

FIG. 2 shows a high level linkage map connecting primary paths in anoverall assembly process in accordance with some embodiments.

FIG. 3 schematically illustrates a barcode assisted assembly process inaccordance with some embodiments.

FIG. 4 schematically illustrates a process for genome assembly inaccordance with some embodiments.

FIG. 5 schematically illustrates aspects of the assembly processes inaccordance with some embodiments.

FIGS. 6A, 6B, 6C, and 6D show de novo assembly of E. coli sequence datausing the barcoded sequencing methods described herein.

FIG. 7 is an example block diagram illustrating a computing device inaccordance with some embodiments.

FIG. 8 illustrates the relationship between the larger contiguousnucleic acid, the different fragments of the larger contiguous nucleicacid, and sequence reads of fragments in accordance with someembodiments.

FIG. 9 illustrates the possible values of h(W) for the sixty-fourpossible k-mers of length three in accordance with some embodiments.

FIG. 10 illustrates the possible k-mers of length three generated fromthe sequence GATCCATCTT in accordance with some embodiments.

FIGS. 11A, 11B, 11C, 11D, and 11E collectively illustrate process flowsfor sequencing in accordance with some embodiments.

FIGS. 12A, 12B, 12C, 12D, 12E, 12F, and 12H collectively illustrate amethod for obtaining a plurality of sequence reads, where the pluralityof sequence reads comprises a plurality of sets of sequence reads, whereeach respective sequence read in a set of sequence reads includes (i) afirst portion that corresponds to a subset of a larger contiguousnucleic acid and (ii) a common second portion that forms an identifierthat is independent of the sequence of the larger contiguous nucleicacid and that identifies a partition, in a plurality of partitions, inwhich the respective sequence read was formed, in accordance with someembodiments.

FIG. 13 illustrates a method for obtaining a plurality of sequence readsand pooling them together in accordance with some embodiments.

FIG. 14 illustrates a de Bruijn graph in accordance with someembodiments.

FIG. 15 illustrates another de Bruijn graph in accordance with someembodiments.

Like reference numerals refer to corresponding parts throughout thepresent disclosure.

DETAILED DESCRIPTION

The present disclosure generally provides methods, processes, andparticularly computer implemented processes and non-transistory computerprogram products for use in the analysis of genetic sequence data. Theprocesses and products described herein are especially useful in theassembly of shorter nucleic acid sequence data into longer linked andpreferably contiguous genetic constructs, including large contigs,chromosomes and whole genomes.

The invention and various specific aspects and embodiments will beunderstood with reference to the following drawings and detaileddescriptions. In some of the drawings and detailed descriptions below,the present invention is described in terms of an important independentembodiment of a system operating on a logic processing device, such as acomputer system. This should not be taken to limit the invention, which,using the teachings provided herein, can be applied to any number oflogic processors working together, whether incorporated into a standalone computer system, an instrument system, or other informationenabled devices or logic components incorporated into laboratory ordiagnostic equipment or in functional communication therewith. Forpurposes of clarity, this discussion refers to devices, methods, andconcepts in terms of specific examples. However, the invention andaspects thereof may have applications to a variety of types of devicesand systems. It is therefore intended that the invention not be limitedexcept as provided in the attached claims.

Furthermore, it is well known in the art that logic systems and methodssuch as described herein can include a variety of different componentsand different functions in a modular fashion. Different embodiments ofthe invention can include different mixtures of elements and functionsand may group various functions as parts of various elements. Forpurposes of clarity, the invention is described in terms of systems thatinclude many different innovative components and innovative combinationsof innovative components and known components. No inference should betaken to limit the invention to combinations containing all of theinnovative components listed in any illustrative embodiment in thisspecification. The functional aspects of the invention that areimplemented on a computer or other logic processing systems or circuits,as will be understood from the teachings herein, may be implemented oraccomplished using any appropriate implementation environment orprogramming language, such as C, C++, Cobol, Pascal, Java, Java-script,HTML, XML, dHTML, assembly or machine code programming, RTL, Python,etc. All references, publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

I. General

In general, assembly of nucleic acid sequence information into longerstretches of contiguous sequence typically involves the identificationof overlapping sequences in different reads, and using those overlappingsequences to map these sequences against each other. Because genomicsequences are very large, the identification of overlapping sequencesonly allows for an inference of linkage between the two sequences. Thisinference is substantially strengthened with larger numbers ofoverlapping sequences in a given region of the overall sequence, e.g.,higher coverage.

In some embodiments, this inference may be further strengthened when thesequences are mapped against a reference sequence. In particular, for anumber of organisms, a reference sequence, e.g., a complete or nearlycomplete sequence of one individual's genome or a consensus of theorganism's genome from multiple individuals, has been elucidated thatprovides a general framework of that organism's genomic sequence. Groupsof overlapping sequences may then generally be mapped against areference sequence for the given organism from which the DNA is beingsequenced in order to provide greater confidence in the identifiedsequence linkage, as well as place the different sequence segments intothe larger genomic context. However, in some embodiments of the presentdisclosure, reference sequences are not used.

In assembling the larger overall sequence, areas in the sequence thathave insufficient information (e.g., insufficient coverage, lack of anunambiguous match to a reference sequence, etc.) can result in breaks inthe overall inferred sequence. These breaks result in a number ofseparate contiguous sequence segments (or contigs) that can requiresubstantial additional processing before they are eventually linked in asingle contiguous sequence. These challenges, while significant insequencing processes where reference sequences are available, are evenmore significant in de novo sequence assembly. By definition, de novoassemblies do not have the benefit of the secondary level ofconfirmation of sequence linkage that comes from a reference sequence.Instead, these sequences must be assembled from the bottom up, e.g., denovo.

In an example process, an aggregation of short sequence reads areobtained from any of a variety of different sequencing processes. Thesequence reads are optionally stored in an appropriate data structurefor further evaluation. A computer algorithm analyzes these sequences toidentify common sequence segments (k-mers) among subsets of thedifferent sequence reads. Identification of k-mer subsequences istypically based upon the level of redundancy or coverage of the k-mersequence of a desired length, which may generally be dependent upon thecomplexity of the genome being sequenced, the depth of sequencingcoverage, and tolerances for varying error types. In many cases, k-merlength may be selected that are anywhere from about 7 to about 31 basesin length, or from about 15 to about 25 bases in length. For example,where a given k-mer is only represented less than 3, 4, 5, 10, 20 ormore times, in the aggregated sequence database, it may be deemedinsufficiently covered to meaningfully be used, depending upon thecomplexity of the sequence being analyzed, etc.

Sequence fragments that include an identified k-mer sequence are thenaligned along that sequence of overlap between the sequences. Additionalk-mer sequence or sequences may then be identified in these overlappingsequences to compile an aggregation of additional sequence segments toextend the contiguous sequence in an initial assembly graph using anadditional assembly algorithm that extends from this k-mer to anoverlapping k-mer to produce a full contig sequence, e.g., using aDeBruijn graph assembler such as Velvet, Euler or the like. Inparticular, the first set of reads having a first k-mer sequence may beused as an anchor for a repeated alignment exercise in which a secondk-mer sequence common to one or more members of the first set, is usedto extend the inferred sequence, i.e., to establish a connection betweenthe first set of short sequences and a second set of short sequences.This is repeated so long as the sequence is able to be extended,unambiguously.

In genomic samples, however, k-mer sequences may be common to multipledisparate portions of the overall sequence, such that the identificationof a subsequent k-mer sequence can implicate two or more different setsof sequence reads as extensions of a given sequence, where those twosequences are not actually related, e.g., one or more of those sequencesis not the correct extension of the sequence. This results in a branchin the inferred connection between sequences, e.g., one of two or moresequences may constitute the continued sequence at this branch point.The maximal unbranched inferred sequence is generally termed a ‘primarypath’. Multiple primary paths are identified for a short read sequencedataset. Different primary paths may then be linked to each otherthrough other processes, e.g., using paired end sequence data,overlaying long read sequence data, or the like.

FIGS. 1A and 1B provide a schematic illustration of the de novo assemblyprocess described above. As shown, sequence fragments 102 are alignedaccording to included k-mer sequences that are represented atsufficiently high levels of coverage, e.g., k-mers 104, 106 and 108, ascompared to the other k-mers that are represented at lower coverage,e.g., k-mers in alignments 120 and 122 k-mers. For example, in somecases, k-mers representing coverages of at least 3-4× would be used foralignment. Although illustrated as k-mers that are only eight bases inlength, this is done for ease of illustration, and actual k-mer lengthsmay vary and may be longer, e.g., 10 mer or longer, 20 mer or longer, 30mer or longer, 40 mer or longer, 50 mer or longer, 60 mer or longer, 70mer or longer, 80 mer or longer, 90 mer or longer, 100 mer or longer.

A series of overlapping k-mer sequences is identified, e.g., k-mers 104and 106, to walk along the aggregate overlapping fragments and assemblea longer contiguous sequence. As shown in FIG. 1B, where a given stepleads to two or more overlapping k-mers, e.g., k-mers 110 and 112, itcreates a branch point 114 in the contiguous sequence. Element 116 ofFIG. 1B illustrates another break point in the contiguous sequence Thestretch of unbranched sequence between the two branch points is definedas a primary path 118. Although illustrated as a relatively shortprimary path, it will be appreciated that longer primary paths aregenerally generated, e.g., comprising 500 bases, 1 kb, 2 kb, 5 kb, ormore in length.

Seed primary paths are then identified from the data. Seed primarypaths, also referred to as seed paths, typically represent primary pathsthat are more likely to have a low copy number within the genome orother large sample nucleic acid (e.g., n approaching 1). This copynumber may be inferred from the length of the sequence in the primarypath, or from the overall read coverage of the primary paths, or both.From each seed path, neighboring or linked primary paths are thenidentified, either through an unambiguous overlap with the seed path, orthrough the use of other linkage information, e.g., long read sequencingdata, or paired end sequence reads that can bridge two primary paths,e.g., one is represented in one primary path while the other end isrepresented in a different primary path. The linkage between two primarypaths may be expressly determined, e.g., through sequencing of theintervening sequence, or it may be an inferred structural linkage wherethe relative position and connection of the two primary paths isinferred, e.g., from paired end sequence reads.

This is shown in FIG. 2, which schematically illustrates a number ofseparate primary paths 202-210, where a structural linkage (shown aslinkages 212) is determined as between the primary paths. As shown, insome cases, linkages will not be determinable.

The longest assembled contiguous sequence segments are then compiled(shown in FIG. 2 as contigs 216 and 218). Typically, lengthy genomefinishing processes, e.g., using laborious Sanger sequencing or otherlong read sequencing processes, e.g., SMRT® sequencing from PacificBiosciences), are then used to close remaining gaps in the overallsequence, e.g., the gap between contigs 206 and 208.

As is clear from the above example, merely assembling a reasonablefinished genome or reasonable number of contigs for a given genome, caninvolve multiple different sequencing processes, e.g., high throughput,short-read shotgun sequencing, paired-end long fragment sequencingand/or low throughput long-read DNA sequencing processes. Each of theseprocesses can be costly and time consuming.

As described herein, however, an improved sequencing and assemblyprocess allows one to obtain substantially more complete assembly fromfewer or even a single, more efficient sequencing processes. Inparticular, in accordance with the processes described herein,structurally linked oligonucleotides that are sequencable using, e.g.,short read sequencing techniques, are provided with common uniqueidentifiers, e.g., barcodes, that may be used in the assembly process toinfer linkage between different sequence segments and/or differentprimary paths, in the assembly process.

II. Linked-Read Sequencing

Barcoding of structurally linked short sequences and resulting sequencereads may be carried out by a number of methods. An exemplary process isdescribed in detail in, for example, U.S. provisional Patent ApplicationNo. 61/940,318, filed Feb. 7, 2014, U.S. provisional Patent ApplicationNo. 61/991,018, filed May 9, 2014, U.S. patent application Ser. No.14/316,383, filed on Jun. 26, 2014, as well as U.S. patent applicationSer. No. 14/175,935, filed Feb. 7, 2014, the full disclosures of whichis hereby incorporated by reference in their entireties.

In brief, sequencing libraries with incorporated barcode sequences areprepared from one or more large nucleic acid sequences, e.g., a genome,chromosome or fragment thereof. This large sample nucleic acid(s) issegmented into first fragments that will typically be on the order of 1to about 100 kb or more in length. These long first fragments are thenpartitioned into separate reaction volumes. While these reaction volumesmay be any of variety of different types of reaction vessels, e.g.,tubes, wells, microwells, nanowells, or the like, in preferred aspects,the reaction volumes (also referred to as partitions), are comprised ofdroplets in an emulsion, e.g., a water-in-oil emulsion).

This partitioning may be carried out so as to ensure that at most, onlya single first fragment is contained in any partition. However, suchprocesses tend to have reduced throughput and higher costs, as thepartitions are inefficiently used where most partitions would remainempty. As such, in preferred aspects, the partitioning is carried outunder conditions in which there are multiple first fragments containedin individual partitions, but where there is a high probability thatwhile multiple first fragments may be present within a given partition,such fragments will be structurally unique, e.g., representingstructurally discrete and disparate regions of the larger sample nucleicacid. Typically, this is accomplished by providing the first fragmentsat a concentration that, based upon the size of the sample nucleic acid,the size of the first fragments, and the reaction volume, willstatistically allocate structurally unique first fragments into eachseparate reaction volume. In particular, such partitioning will resultin fewer than 10% of the partitions having structurally linked separatefirst fragments (overlapping, non-overlapping but immediately adjacent,or adjacent within 0.1 to 5 kb), fewer than 5% of the first fragments ina given partition being structurally linked, fewer than 1% of the firstfragments in a given partition being structurally linked, and in somecases, fewer than 0.5%, 0.2%, or even fewer than 0.1% of the firstfragments in a given partition being structurally linked.

Co-partitioned with the first fragments, e.g., within the same droplet,are collections of oligonucleotides that comprise a common barcodesequence within a given partition. The collection of oligonucleotides istaken from a vast and diverse library of different barcode sequences,such that different partitions have different barcode sequencescontained therein. In preferred aspects, the oligonucleotides comprisingthe barcode sequences are provided into the individual partitionsreleasably attached to a bead or microcapsule, where the barcodesequences included within the oligonucleotides attached to a given beadare all substantially identical. Further, each bead will typicallycomprises more than 100,000, more than 1,000,000, more than 10,000,000and in many cases, more than 50,000,000 individual oligonucleotidemolecules. The oligonucleotides may be drawn from a barcode library thatincludes greater than 1000, greater than 10,000, greater than 100,000,greater than 500,000, greater than 1 million, or even greater than 2million different barcode sequences.

In preferred aspects, these oligonucleotides also include functionalsequences, e.g., amplification primer sequences (which may be targetedto specific sequences, universal priming sequences or random primingsequences), or primer annealing sites, sequencing primer sequences orprimer annealing sites, or the like. In preferred cases, universal orrandom primer sequences are included as at least one of the functionalsequences within the oligonucleotides. These and other functionalsequences are described in, for example, U.S. provisional PatentApplication No. 61/940,318, filed Feb. 7, 2014, U.S. provisional PatentApplication No. 61/991,018, filed May 9, 2014, U.S. patent applicationSer. No. 14/316,383, filed on Jun. 26, 2014, as well as U.S. patentapplication Ser. No. 14/175,935, filed Feb. 7, 2014, the fulldisclosures of which is hereby incorporated by reference in theirentireties.

A population of second, sequencable shorter fragments, that constituteoverlapping fragments of the first fragment, are then generated withinthe reaction volume, where the second short fragments include the commonbarcode sequence segment. In one exemplary process, this is carried outby releasing the oligonucleotides comprising the barcode sequences fromthe beads into the partitions. While the release of the oligonucleotidesmay be carried out using a variety of mechanisms to release theoligonucleotides from the beads, e.g., thermal cleavage, chemicalcleavage, or photo-induced cleavage, in many cases, the release will becarried out by introducing a chemical cleaving agent into the partitionsduring the co-partitioning step that will either cleave a linkagebetween the oligonucleotide and the bead, or degrade the bead such thatthe oligonucleotide is released, or both.

Also co-partitioned with the first fragments and oligonucleotides arereagents for carrying out the amplification of different portions of thefirst fragments, including, for example, DNA polymerase enzyme,nucleoside triphosphates, divalent metal ions, e.g., Mn′ or Mg′.Portions of the first fragments in a given partition are then replicatedusing the oligonucleotides, e.g., including a universal or random primersequence, as primers for that replication, such that different portionsof the first fragment are primed and replicated. In some cases, thesefirst replicate fragments may be additionally primed by theoligonucleotides and replicated. As a result of these replications, acollection of overlapping fragments of the first fragments in a givenpartition is created, where each includes a barcode sequence common tothat partition.

In different partitions, different second fragments are generated fromdifferent first fragments in a similar fashion, with different barcodesequences appended to those second fragments. Across the collection ofdifferent partitions is then created a substantial sequencing library,where elements of that library that are created in the same partition,and thus may be derived from the same first fragment, will include thesame barcode sequence.

The different partitions, representing the sequencing library, are thenpooled and the fragments included therein are sequenced to identify theshort second fragment sequences along with their appended barcodesequences. Because second fragments derived from a given first fragmentwill include a common barcode sequence, the presence of that barcodesequence in a given read, or set of reads provides additional linkageinferences that may be applied throughout the assembly process.

III. Application to Assembly A. Generally

As described previously, a number of different parameters are used inassembly of shorter sequence reads into longer contiguous linkedsequences. These include the presence of overlapping sequence segmentsbetween and among sequence reads that provide an inference that thesequence reads represent overlapping segments of a larger sequencecontext. The ability to provide longer linked shorter sequences, as wellas the ability to provide linkage inferences as between larger sequencefragments from a given nucleic acid, e.g., the ability to provide fewercontigs or even a single unified sequence for the larger molecule, willdepend to some extent upon the level and uniformity of sequencingcoverage for the entire target nucleic acid, where regions with lowerlevels of sequencing coverage may result in gaps between contigs. As aresult, sequencing processes that have more low coverage areas oftenresult in more discontinuous sequence data, e.g., a larger number ofcontigs. While some of the coverage deficiencies can be overcome bybrute force, e.g., sequencing a genome to much greater depth, thatsolution comes at a potentially large cost, in terms of sequencingprocess costs. However, in many cases, coverage deficiencies aresystematic, e.g., resulting in part from the sequence itself.

By contrast, the assembly of barcoded sequencing libraries, as describedherein, can provide enhanced linkage information both at the shortsequence read level as well as in the higher level assembly, withoutnecessarily requiring significant increases in coverage, or the use ofancillary additional sequencing processes.

In particular, in typical short sequence read assembly processes, shortsequence reads that include overlapping sequence segments are alignedwith each other to provide a larger contiguous sequence context, orcontig. These contigs may then be subjected to further assembly withother contigs de novo, or by being mapped against a reference sequence.However, because these sequence reads are extremely short, e.g., 100-200bases in length, in the context of genomes that are in the millions tobillions of bases, the potential for any given read to map uniquely to asingle locus within the genome is relatively low, and this can introducesignificant ambiguity in the assembly process. This ambiguity may belessened by sequencing large numbers of overlapping fragments to providegreater sequencing coverage at a give locus. However, low coverage areasof a genome will still provide difficulties in any assembly process,often leaving gaps between contigs. While lower coverage areas may bebridged by other methods as described elsewhere herein, e.g., long readsequencing, such methods add to the cost and inefficiency of theprocesses.

In the context of the methods described herein, however, short readsequences are provided with barcode sequences that provide additionalinferences of structural relationship between two or more sequence readswithout the requirement of as much sequencing coverage. In particular,aligned short read sequences may be assigned to an aligned contig basedupon both an overlapping sequence with other sequences, as well as basedupon their inclusion of a common barcode sequence. Using the additionalstructural linkage information of an associated common barcode sequenceresults in the identification of much longer contigs.

Likewise, bridging between two contigs may additionally be aided by thepresence of barcode sequences. For example, where adjacent identifiedcontigs include sequence reads that share overlapping barcode sequences,but are otherwise non-overlapping or insufficiently overlapping to aligntogether, one may infer a structural linkage as between those twocontigs. Briefly, the inclusion in two sequence reads provides areasonably strong inference that the two reads were generated fromfragments created within a single partition. When combined withadditional linkage information, e.g., alignment of the sequence read totwo or more contigs, or where two barcodes straddle the same twocontigs, it gives a significant indication of the linkage of those twocontigs.

In terms of an exemplary computer implemented process, a collection ofshort read sequences that include associated barcode sequences, areprovided within a data structure. Subsets of the sequences are alignedwith each other based upon overlapping sequences within those sequences,and also based upon the association of such sequences with the samebarcode sequence. Alignable sequences that do not include the samebarcode sequence, or sequences that include the same barcode sequencesbut that are not alignable are not included, and may be aligned againstdifferent subsets of sequences. Based upon a set of aligned, commonlybarcoded sequences, a linear contiguous sequence, or contig isgenerated. This is repeated with multiple different subsets ofsequences, to provide multiple different contigs. As will beappreciated, the contigs may be longer than what would be achieved basedupon sequence alignment alone, or may be provided with higher levels ofconfidence, despite being based upon lower sequencing coverage.

The generated contigs are then processed further to provide relativeorientation and genomic context with respect to each other to establisha scaffold for the overall genomic (or other genomic component)sequence. In some cases, this may be achieved through mapping of thediscrete contigs against a reference sequence. In many cases, however,mapping against a reference may not be unambiguous, e.g., based upon thepresence of multiple duplicate sequence regions within the genome, orbecause a suitable reference sequence does not exist.

In such cases, barcoded sequences may additionally be used to providelinkage information as between contigs. For example, as noted above, insome cases, sequences that align to a first subset of sequences, butthat do not share a common first barcode sequence, e.g., they include asecond barcode sequence, would not be used in creating the first contig.However, such sequences may be aligned in creating a second contig.Likewise, sequences that include a first barcode, but that align to thesecond contig sequences may not have been used to generate the secondcontig. However, based upon such sequences being present in the datastructure, one can ascertain a linkage between the first and secondcontig. In the context of the processes, contig linkage may beidentified based upon the presence of a threshold level of sharedbarcodes as between sequences that align to different contigs, and/orbased upon the presence of a threshold level of sequences that thatalign to the first contig but include barcodes associated with thesecond contig. As used herein, such threshold level may include a singlesequence or, 2 sequences or more, 3 sequences or more, 4 sequences ormore, 5 sequences or more, 10 sequences or more, or 20 sequences ormore.

B. Application to De Novo Assembly Processes

The barcoded sequencing libraries described above have particularadvantages in de novo assembly. In particular, as noted above, thepresence of a common barcode in two sequence reads can provide aninference of structural linkage between the two sequences, as there isan increased probability that these sequences were created within thesame partition, and from the same first fragment. While this may not bedefinitive, as the same barcode sequence may be present in otherpartitions and in fragments of structurally unrelated first fragments,when combined with other indicators of structural relation, e.g.,overlapping sequences, it becomes a powerful inference of structurallinkage. By way of example, and with reference to the assembly processdescribed above, FIG. 3 schematically illustrates a potential impact ofbarcoded sequences on the assembly processes.

In one aspect, the barcodes included within the sequence reads may beused to provide a structural relationship between and among paths in aninitial assembly in order to provide high level scaffolding of asequence, e.g., providing a high confidence primary path scaffold inwhich the relative order of primary paths, and preferably, highconfidence primary paths is identified and mapped, and against whichfinal genomic assembly may be mapped. As will be appreciated, a highconfidence primary path will generally refer to a primary path that,based upon its length and complexity, will be expected to have a lowprobability of duplication or multiple duplication within a genome,allowing a higher level of confidence that sequences or primary pathsthat connect to that primary path are, in fact, actually linked in thegenome, as opposed to being linked to primary paths having a duplicatesequence. In preferred aspects, the high confidence primary paths areprimary paths that will have a predicted copy number in the relevantgenome approaching 1, e.g., less then 2, or equal to about 1.

In brief, a set of primary paths will have been identified from theinitial assembly graph as described above, including high confidenceprimary paths (also referred to herein as “single copy number”, “lowcopy number”, or “CN1” paths). Each sequence read included within agiven primary path will include an associated barcode sequence, as alsodescribed above. A list of barcodes associated with each primary pathmay then be generated for each primary path. Using the associatedbarcoded sequences, an initial sequence path may be plotted betweenadjacent CN1 primary paths, using those primary paths (both CN1 andotherwise) that share common barcode sequences. The path is thenextended to the next adjacent CN1 path, providing ordering of the CN1paths relative to each other. As will be appreciated, in many cases,duplicate barcode sequences may exist within the overall sequencingprocess and be attached to structurally distinct portions of a largernucleic acid, e.g., chromosomal fragment, or genome fragment. However,because the assembly process focuses on low copy number or CN1 paths,utilizes highly diverse barcode libraries (e.g., greater than 1000,5000, 10,000, 100,000 or even 1 million different barcode sequences),and is addressing very large genetic samples, it would be expected thatthe probability of the same barcode sequence being attached to similaror overlapping sequences that derive from structurally distinct, e.g.,not structurally linked, parts of the genome or genomic fragment, wouldbe extremely low, e.g., in many cases, less than 0.0001%.

This connection process is repeated among the CN1 paths until the orderof all of the CN1 paths is established. The CN1 paths may then be usedas a scaffold for completing assembly of the sequence between the paths.This is generally accomplished in a similar fashion, by using thesequence reads that include barcodes common to the CN1 paths to create alocal sequence assembly graph between adjacent CN1 paths.

In particular, sequence reads including the same barcode sequences as agiven primary path are used to stitch together a local sequence assemblygraph between two adjacent primary paths in the scaffold, in the samefashion as described above for assembly of the initial assembly graph.This assembly map provides the sequence for the space between the twoprimary paths, closing the sequence gap. By utilizing barcodeinformation associated with each sequence in which the k-mer exists, inaddition to the overlapping sequence data in the k-mers, one canunambiguously identify the sequence extending between two primary paths.This is schematically illustrated in FIG. 3. As shown, a series of k-mersequences 302-316, similar to those schematically illustrated for theglobal assembly graph in FIG. 1A are illustrated, except where eachk-mer is coupled to an incorporated barcode sequence 318, e.g., barcodesequence “1”, barcode sequence “2” or barcode sequence “n”. As with theinitial assembly graph, the presence of diverging kmer sequences at agiven point or points could give rise to branch points within thesequence, e.g., branch points 320 and 322. However, by relying in parton the barcode information associated with each k-mer sequence, one caneffectively identify the correct sequence path, eliminating the branchpoint. In particular, with reference to branch point 322, while both ofk-mers 314 and 316 represent possible next sequence steps, because onlythe path associated with k-mer 314 includes the common barcode “1”, itis identified as the next sequence step.

C. Exemplary Embodiments

FIG. 7 is a block diagram illustrating a sequencing system 100 inaccordance with some implementations. The device 100 in someimplementations includes one or more processing units CPU(s) 702 (alsoreferred to as processors), one or more network interfaces 704, a userinterface 706, a memory 712, and one or more communication buses 714 forinterconnecting these components. The communication buses 714 optionallyinclude circuitry (sometimes called a chipset) that interconnects andcontrols communications between system components. The memory 712typically includes high-speed random access memory, such as DRAM, SRAM,DDR RAM, ROM, EEPROM, flash memory, CD-ROM, digital versatile disks(DVD) or other optical storage, magnetic cassettes, magnetic tape,magnetic disk storage or other magnetic storage devices, other randomaccess solid state memory devices, or any other medium which can be usedto store desired information; and optionally includes non-volatilememory, such as one or more magnetic disk storage devices, optical diskstorage devices, flash memory devices, or other non-volatile solid statestorage devices. The memory 712 optionally includes one or more storagedevices remotely located from the CPU(s) 702. The memory 712, oralternatively the non-volatile memory device(s) within the memory 712,comprises a non-transitory computer readable storage medium. In someimplementations, the memory 712 or alternatively the non-transitorycomputer readable storage medium stores the following programs, modulesand data structures, or a subset thereof:

-   -   an operating system 716, which includes procedures for handling        various basic system services and for performing hardware        dependent tasks;    -   a network communication module (or instructions) 718 for        connecting the device 700 with other devices, or a communication        network;    -   a sequence read processing module 720 for processing sequence        reads;    -   an optional representation of a larger contiguous nucleic acid        726 obtained from a biological sample;    -   a plurality of sequence reads 728, each respective sequence read        in the plurality of sequence reads comprising at least a first        portion 730 that corresponds to a subset of the larger        contiguous nucleic acid and a common second portion 732 that        forms an identifier that is independent of the sequence of the        larger contiguous nucleic acid and that identifies a partition,        in a plurality of partitions, in which the respective sequence        read was formed; and    -   a hash table 740 comprising, for each respective sequence read        728, the associated second portion 744 (bar code) and k-mers        obtained by a hash of the respective sequence read 728 according        to a predetermined k-mer length L

In some implementations, the user interface 706 includes an input device(e.g., a keyboard, a mouse, a touchpad, a track pad, and/or a touchscreen) 710 for a user to interact with the system 700 and a display708.

In some implementations, one or more of the above identified elementsare stored in one or more of the previously mentioned memory devices,and correspond to a set of instructions for performing a functiondescribed above. The above identified modules or programs (e.g., sets ofinstructions) need not be implemented as separate software programs,procedures or modules, and thus various subsets of these modules may becombined or otherwise re-arranged in various implementations. In someimplementations, the memory 712 optionally stores a subset of themodules and data structures identified above. Furthermore, in someembodiments, the memory stores additional modules and data structuresnot described above.

Although FIG. 7 shows a “sequence system 700,” FIG. 7 is intended moreas functional description of the various features which may be presentin computer systems than as a structural schematic of theimplementations described herein. In practice, and as recognized bythose of ordinary skill in the art, items shown separately could becombined and some items could be separated.

FIG. 8 illustrates the relationship between the larger contiguousnucleic acid 802, the different fragments 804 of the larger contiguousnucleic acid, and sequence reads 728 of fragments. Typically, between 1and 250 fragments 804, between 5 and 500 fragments 804 or between 10 and1000 fragments 804 are partitioned into a separate partition. In anyevent, sufficiently few of the fragments 804 are partitioned into thesame partition such that the chance that the fragments 804 in a singlepartition have any appreciable overlapping sequences is unlikely.Sequence reads 728 of each fragment 804 are made. In typicalembodiments, sequence reads 728 are short in length (e.g., less than1000 bases) so that they can be sequenced in automated sequencers. Eachsequence read 728 in a partition includes a common second portion 732that forms an identifier that is independent of the sequence of thelarger contiguous nucleic 802 acid nucleic acid and that identifies thepartition, in a plurality of partitions, in which the respectivesequence read was formed.

In some embodiments, a respective set of k-mers for each sequence read728 in the plurality of sequence reads is constructed. In suchembodiments the identifier (barcode 732) of the set of sequence readsthat includes the sequence read is retained for each k-mer in theplurality of k-mers. K-mers are shorter than sequence reads. In someembodiments a sequence read is between 50 and 250 bases whereas a k-merhas a value of k (meaning that it has a nucleic acid length of k) thatis between 7 and 51. Each respective set of k-mers includes at leasteighty percent, at least eight-five percent, at least ninety percent, atleast ninety-five percent of the possible k-mers of the correspondingsequence read.

FIG. 11 is a flow chart illustrating a method (1100) of sequencing alarger contiguous nucleic acid (e.g., obtained from a biological sample)(1102), in accordance with some embodiments. The method is performed ata computer system having one or more processors, and memory storing oneor more programs for execution by the one or more processors (1104).

Obtaining a plurality of sequence reads. In accordance with thedisclosed systems and methods, a plurality of sequence reads 728 isobtained (1106). The plurality of sequence reads comprises a pluralityof sets of sequence reads. Each respective sequence read 728 in a set ofsequence reads comprises a first portion (130) that corresponds to asubset of the larger contiguous nucleic acid and a common second portion(732) that forms an identifier that is independent of the sequence ofthe larger contiguous nucleic acid and that identifies a partition, in aplurality of partitions, in which the respective sequence read wasformed. In other words, the identifier is not derived from, or afunction of the sequencing data of the larger contiguous nucleic acid.

In some embodiments, as illustrated in FIG. 8, to obtain the pluralityof sequence reads 728, a larger contiguous nucleic acid 802 isfragmented to form fragments 804 and these fragments arecompartmentalized, or partitioned into discrete compartments orpartitions (referred to interchangeably herein as partitions). In someembodiments, more than 10, more than 100, more than 1000, more than10,000, more than 100,000, more than 1×10⁶, or more than 5×10⁶ sets ofsequence reads are obtained, corresponding more than 10, more than 100,more than 1000, more than 10,000, more than 100,000, more than 1×10⁶, ormore than 5×10⁶ partitions.

Each partition maintains separation of its own contents from thecontents of other partitions. As used herein, the partitions refer tocontainers or vessels that may include a variety of different forms,e.g., wells, tubes, micro or nanowells, through holes, or the like. Inpreferred aspects, however, the partitions are flowable within fluidstreams. In some embodiments, these vessels are comprised of, e.g.,microcapsules or micro-vesicles that have an outer barrier surroundingan inner fluid center or core, or have a porous matrix that is capableof entraining and/or retaining materials within its matrix. In apreferred aspect, however, these partitions comprise droplets of aqueousfluid within a non-aqueous continuous phase, e.g., an oil phase. Avariety of different suitable vessels are described in, for example,U.S. patent application Ser. No. 13/966,150, filed Aug. 13, 2013, whichis hereby incorporated by reference herein in its entirety. Likewise,emulsion systems for creating stable droplets in non-aqueous or oilcontinuous phases are described in detail in, e.g., Published U.S.Patent Application No. 2010-0105112, which is hereby incorporated byreference herein in its entirety. In certain embodiments, microfluidicchannel networks are particularly suited for generating partitions.Examples of such microfluidic devices include those described in detailin Provisional U.S. Patent Application No. 61/977,804, filed Apr. 4,2014, the full disclosure of which is incorporated herein by referencein its entirety for all purposes. Alternative mechanisms may also beemployed in the partitioning of individual cells, including porousmembranes through which aqueous mixtures of cells are extruded intonon-aqueous fluids. Such systems are generally available from, e.g.,Nanomi, Inc.

In the case of droplets in an emulsion, partitioning the fragments 802into discrete partitions may generally be accomplished by flowing anaqueous, sample containing stream, into a junction into which is alsoflowing a non-aqueous stream of partitioning fluid, e.g., a fluorinatedoil, such that aqueous droplets are created within the flowing streampartitioning fluid, where such droplets include the sample materials. Asdescribed below, the partitions, e.g., droplets, also typically includeco-partitioned barcode oligonucleotides.

The relative amount of sample materials within any particular partitionmay be adjusted by controlling a variety of different parameters of thesystem, including, for example, the concentration of fragments 804 inthe aqueous stream, the flow rate of the aqueous stream and/or thenon-aqueous stream, and the like. The partitions described herein areoften characterized by having overall volumes that are less than 1000pL, less than 900 pL, less than 800 pL, less than 700 pL, less than 600pL, less than 500 pL, less than 400 pL, less than 300 pL, less than 200pL, less than 100 pL, less than 50 pL, less than 20 pL, less than 10 pL,or even less than 1 pL. Where co-partitioned with beads, it will beappreciated that the sample fluid volume within the partitions may beless than 90% of the above described volumes, less than 80%, less than70%, less than 60%, less than 50%, less than 40%, less than 30%, lessthan 20%, or even less than 10% of the above described volumes. In somecases, the use of low reaction volume partitions is particularlyadvantageous in performing reactions with small amounts of startingreagents, e.g., input larger contiguous nucleic acid fragments. Methodsand systems for analyzing samples with low input nucleic acids arepresented in U.S. Provisional Patent Application No. 62/017,580 Jun. 26,2014, the full disclosure of which is hereby incorporated by referencein its entirety.

Once the fragments 804 are introduced into their respective partitions,the fragments 804 within partitions are generally provided with uniqueidentifiers such that, upon characterization of those fragments 804,they may be attributed as having been derived from their respectivepartitions. In some embodiments, such unique identifiers are previously,subsequently or concurrently delivered to the partitions that hold thecompartmentalized or partitioned fragments 804, in order to allow forthe later attribution of the characteristics, e.g., nucleic acidsequence information, to the sample nucleic acids included within aparticular compartment, and particularly to relatively long stretches ofcontiguous sample nucleic acids that may be originally deposited intothe partitions.

Accordingly, the fragments 804 are typically co-partitioned with theunique identifiers 732 (e.g., barcode sequences). In particularlypreferred aspects, the unique identifiers 732 are provided in the formof oligonucleotides that comprise nucleic acid barcode sequences 732.The oligonucleotides 732 are partitioned such that as betweenoligonucleotides in a given partition, the nucleic acid barcodesequences 732 contained therein are the same, but as between differentpartitions, the oligonucleotides can, and preferably have differingbarcode sequences. In preferred embodiments, only one nucleic acidbarcode sequence 732 is associated with a given partition, although insome embodiments, two or more different barcode sequences are present ina given partition.

The larger contiguous nucleic acid 802 is typically fragmented andpartitioned such that the fragments 804 of the larger contiguous nucleicacid 802 in the partitions are long fragments or stretches of contiguousnucleic acid molecules. As illustrated in FIG. 8, these fragments 804typically represent a number of overlapping fragments of the overalllarger contiguous nucleic acid to be analyzed, e.g., an entirechromosome, exome, or other large genomic fragment.

The larger contiguous nucleic acid 802 is also typically fragmented andpartitioned at a level whereby a given partition has a very lowprobability of including two overlapping fragments 804 of the startinglarger contiguous nucleic acid 802. This is typically accomplished byproviding the larger contiguous nucleic acid 802 at a low input amountand/or concentration during the partitioning process. As a result, inpreferred cases, a given partition includes a number of long, butnon-overlapping fragments 804 of the starting larger contiguous nucleicacid. The nucleic acid fragments in the different partitions are thenassociated with unique identifiers 732 where, for any given partition,fragments 804 contained therein possess the same unique identifier, butwhere different partitions include different unique identifiers.Moreover, because the partitioning step allocates the sample componentsinto very small volume partitions or droplets, it will be appreciatedthat in order to achieve the desired allocation as set forth above, oneneed not conduct substantial dilution of the sample, as would berequired in higher volume processes, e.g., in tubes, or wells of amultiwell plate. Further, because the systems described herein employsuch high levels of barcode diversity, one can allocate diverse barcodesamong higher numbers of genomic equivalents, as provided above. In someembodiments, in excess of 10,000, 100,000, 500,000, etc. diverse barcodetypes are used to achieve genome:(barcode type) ratios that are on theorder of 1:50 or less, 1:100 or less, 1:1000 or less, or even smallerratios, while also allowing for loading higher numbers of genomes (e.g.,on the order of greater than 100 genomes per assay, greater than 500genomes per assay, 1000 genomes per assay, or even more) while stillproviding for far improved barcode diversity per genome. Here, each suchgenome is an example of a larger contiguous nucleic acid.

Referring to FIG. 12A, panel B, often the above-described partitioningis performed by combining the sample containing the larger contiguousnucleic acid 802 with a set of oligonucleotide tags 1202 (containing thebarcodes 732) that are releasably-attached to beads prior to thepartitioning step. The oligonucleotides 1202 may comprise at least aprimer region 1210 and a barcode 732 region. Between oligonucleotides1202 within a given partition, the barcode region 732 is substantiallythe same barcode sequence, but as between different partitions, thebarcode region 732 in most cases is a different barcode sequence. Insome embodiments, the primer region includes (or further comprises) anN-mer 1216 (either a random N-mer or an N-mer designed to target aparticular sequence) that is used to prime the fragments 804 within thepartitions. In some cases, where the N-mer is designed to target aparticular nucleic acid sequence, the primer region 1210 is designed totarget a particular chromosome (e.g., human chromosome 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, or Y),or region of a chromosome, e.g., an exome or other targeted region. Insome cases, the N-mer is designed to target a particular gene or geneticregion, such as a gene or region associated with a disease or disorder(e.g., cancer). In some cases, the N-mer is designed to target aparticular structural variation. Within the partitions, an amplificationreaction is conducted using the primer sequence 1210/1216 (e.g. N-mer)to prime the fragments 804 at different places along their lengths. As aresult of the amplification, each partition contains amplified productsof the fragments 804, termed sequence reads 728, that comprise anidentical or near-identical (common) barcode 732, and that representoverlapping, smaller fragments of the fragments 804 in each partition.The barcode 732 therefore serves as a marker that signifies that a setof sequence reads 728 originated from the same partition, and thuspotentially also originated from the same fragment 804. It will beappreciated that there are typically several fragments 804 in any givenpartition. Nevertheless, in typical embodiments, fragments 804 that arein the same partition typically do not have any significant overlap andso it is possible to localize the amplified sequence reads 728 to thecorrect fragment 804 in any given partition. Following amplification,the sequence reads 728 are pooled, sequenced, and aligned using asequencing algorithm. Because shorter sequence reads 728 may, by virtueof their associated barcode sequences 732, be aligned and attributed toa single, long fragment 804 of the larger contiguous nucleic acid 802,all of the identified variants on that sequence can be attributed to asingle originating fragment 802 and single originating chromosome of thelarger contiguous nucleic acid. Further, by aligning multiple co-locatedvariants across multiple fragments 804, one can further characterizethat chromosomal contribution. Accordingly, conclusions regarding thephasing of particular genetic variants may then be drawn. Suchinformation may be useful for identifying haplotypes, which aregenerally a specified set of genetic variants that reside on the samenucleic acid strand or on different nucleic acid strands. Moreover,additionally or alternatively, structural variants are identified.

In some embodiments, referring to FIG. 12A, the co-partitionedoligonucleotides 1202 also comprise functional sequences in addition tothe barcode sequence 732 and the primer region sequence 1210/1216. Forinstance, in some embodiments, the co-partitioned oligonucleotides 1202also comprise other functional sequences useful in the processing of thepartitioned nucleic acids such as targeted or random/universalamplification primer sequences for amplifying the fragments 804 withinthe partitions while attaching the associated barcode sequences,sequencing primers, hybridization or probing sequences, e.g., foridentification of presence of the sequences, or for pulling downbarcoded nucleic acids, or any of a number of other potential functionalsequences. See, for example, the disclosure on co-partitioning ofoligonucleotides and associated barcodes and other functional sequences,along with sample materials as described in, for example, U.S. PatentApplication Nos. 61/940,318, filed Feb. 7, 2014, 61/991,018, Filed May9, 2014, and U.S. patent application Ser. No. 14/316,383, filed on Jun.26, 2014, as well as U.S. patent application Ser. No. 14/175,935, filedFeb. 7, 2014, the full disclosures of which is hereby incorporated byreference in their entireties.

In one exemplary process, beads are provided that each includes largenumbers of the above described oligonucleotides 1202 releasably attachedto the beads, where all of the oligonucleotides attached to a particularbead include the same nucleic acid barcode sequence 732, but where alarge number of diverse barcode sequences are represented across thepopulation of beads used. Typically, the population of beads provides adiverse barcode sequence library that includes at least 1000 differentbarcode sequences, at least 10,000 different barcode sequences, at least100,000 different barcode sequences, or in some cases, at least1,000,000 different barcode sequences. Additionally, each bead typicallyis provided with large numbers of oligonucleotide molecules 1202attached. In particular, the number of molecules of oligonucleotides1202 including the barcode sequence 732 on an individual bead may be atleast about 10,000 oligonucleotides, at least 100,000 oligonucleotidemolecules, at least 1,000,000 oligonucleotide molecules, at least100,000,000 oligonucleotide molecules, and in some cases at least 1billion oligonucleotide molecules.

In some embodiments, the oligonucleotides are releasable from the beadsupon the application of a particular stimulus to the beads. In somecases, the stimulus is a photo-stimulus, e.g., through cleavage of aphoto-labile linkage that may release the oligonucleotides. In somecases, a thermal stimulus is used, where elevation of the temperature ofthe beads environment results in cleavage of a linkage or other releaseof the oligonucleotides form the beads. In some cases, a chemicalstimulus is used that cleaves a linkage of the oligonucleotides to thebeads, or otherwise results in release of the oligonucleotides from thebeads.

In some embodiments, the beads including the attached oligonucleotides1202 are co-partitioned with the individual samples, such that a singlebead and a single sample are contained within an individual partition.In some cases, where single bead partitions are desired, it may bedesirable to control the relative flow rates of the fluids such that, onaverage, the partitions contain less than one bead per partition, inorder to ensure that those partitions that are occupied, are primarilysingly occupied. Likewise, in some embodiments, the flow rate iscontrolled to provide that a higher percentage of partitions areoccupied, e.g., allowing for only a small percentage of unoccupiedpartitions. In preferred aspects, the flows and channel architecturesare controlled as to ensure a desired number of singly occupiedpartitions, less than a certain level of unoccupied partitions and lessthan a certain level of multiply occupied partitions.

FIG. 3 of U.S. Patent Application No. 62/072,214, filed Oct. 29, 2014,entitled “Analysis of Nucleic Acid Sequences,” which is herebyincorporated by reference and the portions of the specificationdescribing FIG. 3 provide a detailed example of one method for barcodingand subsequently sequencing a larger contiguous nucleic acid (referredto in the reference as a “sample nucleic acid”) in accordance with oneembodiment of the present disclosure. As noted above, while single beadoccupancy may be the most desired state, it will be appreciated thatmultiply occupied partitions, or unoccupied partitions may often bepresent. FIG. 4 of U.S. Patent Application No. 62/072,214, filed Oct.29, 2014, entitled “Analysis of Nucleic Acid Sequences,” which is herebyincorporated by reference, and the portions of the specificationdescribing FIG. 4 provide a detailed example of a microfluidic channelstructure for co-partitioning samples and beads comprising barcodeoligonucleotides in accordance with one embodiment of the presentdisclosure.

Once co-partitioned, the oligonucleotides 1202 disposed upon the beadare used to barcode and amplify the partitioned samples. One process foruse of these barcoded oligonucleotides 1202 in amplifying and barcodingsamples is described in detail in U.S. Patent Application Nos.61/940,318, filed Feb. 7, 2014, 61/991,018, filed May 9, 2014, and Ser.No. 14/316,383, (Attorney Docket No. 43487-708.201) filed on Jun. 26,2014, the full disclosures of which are hereby incorporated by referencein their entireties. Briefly, in one aspect, the oligonucleotidespresent on the beads that are co-partitioned with the samples arereleased from their beads into the partition with the samples. Theoligonucleotides typically include, along with the barcode sequence 732,a primer sequence at its 5′ end 1216. In some embodiments, this primersequence is a random oligonucleotide sequence intended to randomly primenumerous different regions of the samples. In some embodiments theprimer sequence 1216 is a specific primer sequence targeted to primeupstream of a specific targeted region of the sample.

Once released, the primer portion 1216 of the oligonucleotide 1202anneals to a complementary region of fragments 804 in the partition.Extension reaction reagents, e.g., DNA polymerase, nucleosidetriphosphates, co-factors (e.g., Mg²⁺ or Mn²⁺ etc.), that are alsoco-partitioned with the fragments 804 and beads, extend the primersequence using the fragments 804 as a template, to produce acomplementary sequence to the strand of the fragment 804 to which theprimer annealed, and this complementary sequence includes theoligonucleotide 1202 and its associated barcode sequence 732. Annealingand extension of multiple primers to different portions of the fragments804 in the partition may result in a large pool of overlappingcomplementary portions of the fragments 804, each possessing its ownbarcode sequence 732 indicative of the partition in which it wascreated. In some cases, these complementary portions may themselves beused as a template primed by the oligonucleotides 1202 present in thepartition to produce a complement of the complement that again, includesthe barcode sequence 732. In some cases, this replication process isconfigured such that when the first complement is duplicated, itproduces two complementary sequences at or near its termini, to allowthe formation of a hairpin structure or partial hairpin structure thatreduces the ability of the molecule to be the basis for producingfurther iterative copies. A schematic illustration of one example ofthis is shown in FIG. 12.

As FIG. 12A shows, oligonucleotides 1202 that include a barcode sequence732 are co-partitioned in, e.g., a droplet 1204 in an emulsion, alongwith a sample fragment 306. In some embodiments, the oligonucleotides302 are provided on a bead 1208 that is co-partitioned with the largercontiguous nucleic acid fragment 804. The oligonucleotides 1202 arepreferably releasable from the bead 1208, as shown in FIG. 12A, panel(A). As shown in FIG. 12A panel (B), the oligonucleotides 1202 includesa barcode sequence 732, in addition to one or more functional sequences,e.g., sequences 1212, 732, 1210, and 1216. For example, theoligonucleotide 1202 is shown as further comprising sequence 1212 thatmay function as an attachment or immobilization sequence for a givensequencing system, e.g., a P5 sequence used for attachment in flow cellsof an ILLUMINA, HISEQ or MISEQ system. In other words, attachmentsequence 1212 is used to reversibly attach oligonucleotide 1202 to abead 1208 in some embodiments. As shown in FIG. 12A, panel B, theoligonucleotide 1202 also includes a primer sequence 1210, which mayinclude (or further comprise) a random or targeted N-mer 1216 (discussedabove) for priming replication of portions of the fragment 804. Alsoincluded within exemplary oligonucleotide 1202 of FIG. 12A, panel B, isa sequence 1210 which may provide a sequencing priming region, such as a“read1” or R1 priming region, that is used to prime polymerase mediated,template directed sequencing by synthesis reactions in sequencingsystems. In many cases, the barcode sequence 732, and immobilizationsequence 1212 may be common to all of the oligonucleotides 1202 attachedto a given bead. The primer sequence 1210/1216 may vary for random N-merprimers, or may be common to the oligonucleotides on a given bead forcertain targeted applications.

Referring to FIG. 12B, based upon the presence of primer sequence 1216,the oligonucleotides 1202 a and 1202 b are able to prime the fragment804, which allows for extension of the oligonucleotides 1202 a and 1202b using polymerase enzymes and other extension reagents alsoco-portioned with the bead 1208 and fragment 804.

As shown in FIG. 12C, following extension of the oligonucleotides that,for random N-mer primers, would anneal to multiple different regions ofthe fragment 804; multiple overlapping complements of the fragment 804are created, e.g., sequence reads 728-1 and 728-2. As such, FIG. 12Cillustrates (A) obtaining a plurality of sequence reads, where eachrespective sequence read 728 in the plurality of sequence readscomprises a first portion 730 that corresponds to a subset of the largercontiguous nucleic acid 802 and a common second portion 1250 that thatforms an identifier 732 that is independent of the sequence of thelarger contiguous nucleic acid and that identifies a partition, in aplurality of partitions, in which the respective sequence read 728 wasformed (e.g., bar code 732).

The barcoded sequence reads 728 of FIG. 12B may then be subjected tocharacterization, e.g., through sequence analysis, or they may befurther amplified in the process, as shown in FIG. 12D. For example,additional oligonucleotides, e.g., oligonucleotide 1202 c, also releasedfrom bead 1208, may prime the fragment 1202 b. In particular, again,based upon the presence of the random N-mer primer 1216 inoligonucleotide 1202 c (which in many cases will be different from otherrandom N-mers in a given partition) the oligonucleotide anneals with thefragment 1202 b, and is extended to create a complement 728-3 to atleast a portion of fragment 1202 b which comprises a duplicate of aportion of the larger contiguous nucleic acid sequence. Extension of theoligonucleotide 1202 b continues until it has replicated through theoligonucleotide portion 730 of fragment 1202 b. As illustrated in FIG.12D, the oligonucleotides may be configured to promptly stop in thereplication by the polymerase at a desired point, e.g., afterreplicating through sequences 1216 and 1210 of oligonucleotide 1202 b.As described herein, this may be accomplished by different methods,including, for example, the incorporation of different nucleotidesand/or nucleotide analogues that are not capable of being processed bythe polymerase enzyme used. For example, this may include the inclusionof uracil containing nucleotides within the sequence region 1210 toprevent a non-uracil tolerant polymerase to cease replication of thatregion. As a result, referring to FIG. 12E, a sequence read 728-3 iscreated that includes the full-length oligonucleotide 1202 at one end,including the barcode sequence 732, the attachment sequence 1212, theprimer region 1210, and the random N-mer sequence 1216. At the other endof the sequence is included the complement 1216′ to the random N-mer ofthe first oligonucleotide 1202, as well as a complement to all or aportion of the primer sequence, shown as sequence 1210′. The R1 sequence1210 and its complement 1210′ are then able to hybridize together toform a partial hairpin structure 1260. As will be appreciated, becausethe random N-mers differ among different oligonucleotides, thesesequences and their complements would not be expected to participate inhairpin formation, e.g., sequence 1216′, which is the complement torandom N-mer 1216, would not be expected to be complementary to randomN-mer sequence 1216 b. This would not be the case for otherapplications, e.g., targeted primers, where the N-mers would be commonamong oligonucleotides within a given partition.

By forming these partial hairpin structures, it allows for the removalof first level duplicates of the sample sequence from furtherreplication, e.g., preventing iterative copying of copies. The partialhairpin structure also provides a useful structure for subsequentprocessing of the created fragments, e.g., fragment 730-3.

All of the sequence reads 728 from multiple different partitions maythen be pooled for sequencing on high throughput sequencers as describedherein. Because each sequence read 728 is coded as to its partition oforigin, the sequence of that sequence read 728 may be attributed back toits origin based upon the presence of the barcode 732.

This is schematically illustrated in FIG. 13. As shown in one example, afragment 804-1 and a fragment 804-2 are each partitioned along withtheir own sets of barcode oligonucleotides 1202 as described above.Within each partition, each fragment (804-1 and 804-2) is then processedto separately to provide overlapping sequence reads 728 of the fragments804-1 and 804-2, to form set of sequence reads 1302-1 and 1302-2. Thisprocessing provides sequence reads 728 with a barcode sequence 732 thatis the same for each of the sequence reads 728 derived from a particularfirst fragment 804. As shown, the set of sequence reads 1302-1 isdenoted by “1” while the set of sequence reads 1302-2 is denoted by “2”.A diverse library of barcodes may be used to differentially barcodelarge numbers of different sets of sequence reads. However, it is notnecessary for every sequence read in a given partition to be barcodedwith different barcode sequence. In fact, in many cases, multipledifferent fragments 804 may be processed concurrently in the samepartition to include the same barcode sequence 732. Diverse barcodelibraries are described in detail elsewhere herein.

The sets of sequence reads may then be pooled for sequencing using, forexample, sequence by synthesis technologies available from Illumina orIon Torrent division of Thermo Fisher, Inc. Once sequenced, the sequencereads 728 can be attributed to their respective fragment 804 set, e.g.,as shown in aggregated reads 1314 and 1316, at least in part based uponthe included barcodes, and optionally, and preferably, in part basedupon the sequence of the fragment itself. The attributed sequence readsfor each fragment set are then assembled to provide the assembledsequence for each sample fragment, e.g., sequences 1318 and 1320, whichin turn, may be further attributed back to their respective originalfragments 804. Methods and systems for assembling genomic sequences aredescribed in, for example, U.S. Provisional Patent Application No.62/017,589 (Attorney Docket No. 43487-729.101), filed Jun. 26, 2014, thefull disclosure of which is hereby incorporated by reference in itsentirety.

Referring to FIG. 11A (1108), in some embodiments, a partition in theplurality of partitions comprises at least 10 molecules, at least 100molecules, at least 1000 molecules, at least 1×10⁴ molecules, at least1×10⁵ molecules, at least 1×10⁶ molecules, at least 1×10⁷ molecules, orat least 1×10⁸ molecules with the common second portion 732. In otherwords, the partition includes at least at least 10 molecules, at least100 molecules, at least 1000 molecules, at least 1×10⁴ molecules, atleast 1×10⁵ molecules, at least 1×10⁶ molecules, at least 1×10⁷molecules, or at least 1×10⁸ molecules 1202 (FIG. 12A), and each suchmolecule 1202 further comprises a primer sequence 1210 complementary toat least a portion of the larger contiguous nucleic acid 802.

Referring to FIG. 11A (1110), in some embodiments, a partition in theplurality of partitions comprises at least 10 molecules, at least 100molecules, at least 1000 molecules, at least 1×10⁴ molecules, at least1×10⁵ molecules, at least 1×10⁶ molecules, at least 1×10⁷ molecules, orat least 1×10⁸ molecules with the common second portion 732, and eachsuch molecule 1202 further comprises a primer site 1210 and asemi-random N-mer priming sequence 1216 that is complementary to part ofthe larger contiguous nucleic acid (1110).

Referring to FIG. 11A (1112), in some embodiments, each fragment 804 inthe one or more fragments of the larger contiguous nucleic acid 802 in apartition in the plurality of partitions is greater than 1 kilobase inlength, is greater than 5 kilobases in length, is greater than 50kilobases in length, is greater than 100 kilobases in length, is greaterthan 200 kilobases in length, is greater than 250 kilobases in length,is greater than 300 kilobases in length, or is greater than 350kilobases in length. Typically, the fragments 804 of the largercontiguous nucleic acid 802 that are partitioned into partitions arelonger than 1 kb, longer than 5 kb, longer than 10 kb, longer than 15kb, longer than 20 kb, longer than 30 kb, longer than 40 kb, longer than50 kb, longer than 60 kb, longer than 70 kb, longer than 80 kb, longerthan 90 kb or even longer than 100 kb. In some embodiments the one orfragments in a partition in the plurality of partitions have an N50(where the sum of the fragment 804 lengths that are greater than thestated N50 number is 50% of the sum of all fragment 804 lengths) of atleast about 10 kb, at least about 20 kb, at least about 50 kb, at leastabout 100 kb, at least about 150 kb, at least about 200 kb, at leastabout 250 kb, at least about 300 kb, at least about 350 kb, at leastabout 400 kb, at least about 500 kb, in excess of 1 Mb, or even inexcess of 2 Mb. In some embodiments the one or fragments in eachpartition across the plurality of partitions have an overall N50 (wherethe sum of the fragment 804 lengths that are greater than the stated N50number is 50% of the sum of all fragment 804 lengths) of at least about10 kb, at least about 20 kb, at least about 50 kb, at least about 100kb, at least about 150 kb, at least about 200 kb, at least about 250 kb,at least about 300 kb, at least about 350 kb, at least about 400 kb, atleast about 500 kb, in excess of 1 Mb, or even in excess of 2 Mb. Insome embodiments, the one or more fragments 804 of the larger contiguousnucleic acid 802 in a partition in the plurality of partitions arebetween 20 kilobases and 200 kilobases in length (1114). In someembodiments, the one or more fragments of the larger contiguous nucleicacid in a partition in the plurality of partitions are between 20kilobases and 200 kilobases in length (1116).

Referring to FIG. 11B (1118), in some embodiments, the one or morefragments 804 of the larger contiguous nucleic acid in a partition inthe plurality of partitions consists of between 1 and 500 differentfragments, between 10 and 1000 different fragments, between 100 and 1000different fragments, between 25 and 150 different fragments, between 5and 500 different fragments, between 25 and 400 different fragments,between 100 and 10,000 different fragments, between 50 and 250 differentfragments, or between 2 and 125 different fragments of the largercontiguous nucleic acid (1118). In some embodiments, the one or morefragments 804 of the larger contiguous nucleic acid in a partition inthe plurality of partitions comprises more than 2 different fragments804, more than 10 different fragments 804, more than 20 differentfragments 804, more than 50 different fragments 804, more than 100different fragments 804, more than 200 different fragments 804, morethan 300 different fragments 804, more than 400 different fragments 804,or more than 500 different fragments 804 of the larger contiguousnucleic acid (1118). In a specific embodiment, the one or more fragments804 of the larger contiguous nucleic acid 802 in a partition in theplurality of partitions consists of between 5 and 100 fragments 804 ofthe larger contiguous nucleic acid 802 (1120).

Referring to FIG. 11B (1122), in some embodiments, the plurality ofsequence reads is obtained from less than 5 nanograms of nucleic orribonucleic acid. That is, the total amount of nucleic acid 804 neededfor the plurality of partitions is advantageously less than 5 nanogramsin some embodiments. In varying embodiments in accordance with thepresent disclosure, the plurality of sequence reads is obtained fromless than 500 nanograms of nucleic or ribonucleic acid, less than 250nanograms of nucleic or ribonucleic acid, less than 50 nanograms ofnucleic or ribonucleic acid, less than 5 nanograms of nucleic orribonucleic acid, or less than 2 nanograms of nucleic or ribonucleicacid.

Referring to FIG. 11B (1124), in some embodiments, the identifier in thesecond portion 732 of each respective sequence read 728 in the set ofsequence reads encodes a common value selected from the set {1, . . . ,1024}, the set {1, . . . , 4096}, the set {1, . . . , 16384}, the set{1, . . . , 65536}, the set {1, . . . , 262144}, the set {1, . . . ,1048576}, the set {1, . . . , 4194304}, the set {1, . . . , 16777216},the set {1, . . . , 67108864}, or the set {1, . . . , 1×10¹²}. Forinstance, consider the case in which the identifier is represented by aset of five nucleotide positions. In this instance, each nucleotideposition contributes four possibilities (A, T, C or G), giving rise,when all five positions are considered, to 4×4×4×4×4=1024 possibilities.As such, the five nucleotide positions form the basis of the set {1, . .. , 1024}. Thus, when the barcode sequence is a 5-mer, the secondportion 732 of each sequencing read 728 encodes a unique predeterminedvalue selected from the set {1, . . . , 1024}. Likewise, when thebarcode sequence is represented by a set of six nucleotide positions,the six nucleotide positions collectively contribute 4×4×4×4×4×4=4096possibilities. As such, the six nucleotide positions form the basis ofthe set {1, . . . , 4096}. In other words, when the barcode sequence isa 6-mer, the second portion 732 of each sequencing read 128 encodes aunique predetermined value selected from the set {1, . . . , 4096}. Insome embodiments, the identifier is an N-mer, and N is an integerselected from the set {4, . . . , 20} (1126).

In some embodiments, the identifier 732 of a sequencing read 728 in theplurality of sequencing reads is localized to a contiguous set ofoligonucleotides within the sequencing read. In one such exemplaryembodiment, the contiguous set of oligonucleotides is an N-mer, where Nis an integer selected from the set {4, . . . , 20} (214). In otherwords, in some embodiments, the barcode 732 in, for instance FIG. 12A,panel B, is a contiguous set of nucleotide positions (e.g., 4 contiguousnucleotide positions, 5 contiguous nucleotide positions, 6 contiguousnucleotide positions, 7 contiguous nucleotide positions, 8 contiguousnucleotide positions, 9 contiguous nucleotide positions, 10 contiguousnucleotide positions, 11 contiguous nucleotide positions, 12 contiguousnucleotide positions, 13 contiguous nucleotide positions, 14 contiguousnucleotide positions, 15 contiguous nucleotide positions, 16 contiguousnucleotide positions, 17 contiguous nucleotide positions, 18 contiguousnucleotide positions, 19 contiguous nucleotide positions, or 20contiguous nucleotide positions) within oligonucleotide tag 1202 whichultimately becomes second portion 732 upon transcription of the largercontiguous nucleic acid.

By contrast, in some embodiments, the identifier in the second portionof a sequencing read in the plurality of sequencing reads is localizedto a noncontiguous set of oligonucleotides within the sequencing read.In one such exemplary embodiment, the predetermined noncontiguous set ofnucleotides collectively consists of N nucleotides, where N is aninteger in the set {4, . . . , 20}. As an example, in some embodiments,referring to FIG. 12A, panel B, barcode sequence 732 comprises a firstset of contiguous nucleotide positions at a first position inoligonucleotide tag 1202 and a second set of contiguous nucleotidepositions at a second position in oligonucleotide tag 120, that isdisplaced from the first set of contiguous nucleotide positions by aspacer. In one specific example, the barcode sequence 732 comprises(X1)_(n)Y_(z)(X2)_(m), where X1 is n contiguous nucleotide positions, Yis a constant predetermined set of z contiguous nucleotide positions,and X2 is m contiguous nucleotide positions. In this example, theidentifier in the second portion of the sequencing read 728 produced bya schema invoking this exemplary barcode is localized to a noncontiguousset of oligonucleotides, namely (X1)_(n) and (X2)_(m). This is just oneof many examples of noncontiguous formats for barcode sequence withinthe scope of the present disclosure.

The nucleic acid barcode sequences 732 will typically include from 6 toabout 20 or more nucleotides within the sequence of theoligonucleotides. In some embodiments, these nucleotides are completelycontiguous, e.g., in a single stretch of adjacent nucleotides. Inalternative embodiments, they are separated into two or more separatesubsequences that are separated by one or more nucleotides. Typically,separated subsequences are separated by about 4 to about 16 interveningnucleotides.

Referring to FIG. 11B (1128 and 1130), in some embodiments, an averagesequence read length of the plurality of sequence reads is between 40bases and 200 bases (1128), between 40 bases and 200 bases, between 60bases and 140 bases, or between 30 bases and 130 bases. In someembodiments the sequence reads have a sequence length that is compatiblewith the flow cells of ILLUMINA, HISEQ, MISEQ or related systems.

In some embodiments, the plurality of sequence reads, in the aggregate,have an N50 (where the sum of the sequence read lengths that are greaterthan the stated N50 number is 50% of the sum of all sequence readlengths) of at least about 10 kb, at least about 20 kb, or at leastabout 50 kb. In some aspects, sequence read lengths having an N50 of atleast about 100 kb, at least about 150 kb, at least about 200 kb, and inmany cases, at least about 250 kb, at least about 300 kb, at least about350 kb, at least about 400 kb, and in some cases, or at least about 500kb or more, are attained. In still other cases, maximum sequence readlengths in excess of 200 kb, in excess of 300 kb, in excess of 400 kb,in excess of 500 kb, in excess of 1 Mb, or even in excess of 2 Mb areobtained in accordance with the present disclosure.

Referring to FIG. 11C (1128 and 1130), in some embodiments, a set ofsequence reads in the plurality of sequence reads comprises more than100 sequence reads, and each sequence read 728 of the more than 100sequence reads includes the same common second portion (1132). That is,a given partition in the plurality of partitions yields 100 sequencereads and each such sequence read 728 has the same identifier 732. Moregenerally, in some embodiments, a set of sequence reads in the pluralityof sequence reads comprises more than 10 sequence reads, more than 200sequence reads, more than 500 sequence reads, more than 1000 sequencereads, more than 2500 sequence reads, or more than 5000 sequence readsand each such sequence read 728 includes the same common second portion(the same identifier 732).

Referring to FIG. 11C (1134, 1136), in some embodiments, the largercontiguous nucleic acid 802 is a chromosome (1134). In some embodiments,the larger contiguous nucleic acid 802 is the genome of amulti-chromosomal organism such as a human. In some embodiments, thelarger contiguous nucleic acid 802 includes whole genomes, individualchromosomes, exomes, amplicons, or any of a variety of different nucleicacids of interest. In some embodiments, the larger contiguous nucleicacid 802 is greater than 40 million base pairs in length (1136). In someembodiments, the larger contiguous nucleic acid 802 is greater than100,000 base pairs in length, is greater than 1 million base pairs inlength, is greater than 5 million base pairs in length, is greater than10 million base pairs in length, is greater than 20 million base pairsin length, is greater than 30 million base pairs in length, or isgreater than 50 million base pairs in length.

Create a set of k-mers. Referring to FIG. 11C (1138), once the pluralityof sequence reads is obtained, a respective set of k-mers is created foreach sequence read 728 in the plurality of sequence reads. Takentogether, the sets of k-mers collectively comprise a plurality ofk-mers. That is, the sets of k-mers from the plurality of sequence readsare pooled to form a plurality of k-mers. Importantly, the identifier732 of the source sequence read 728 for each k-mer 746 in the pluralityof k-mers is retained. For instance, a data structure that lists, foreach respective k-mer 746 observed, the barcodes (second portions) 732of the sequence reads 728 that contain the respective k-mer is used insome embodiments of the present disclosure. In general, any technique ordata structure that tracks the barcodes 732 associated with the sequencereads 728 that contain a respective k-mer 746 are within the scope ofthe present disclosure.

The value k is necessarily less than the average length of the sequencereads 728 in the plurality of sequence reads. In some embodiments, khave a value of 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, or 43. In some embodiments k is an eveninteger between 6 and 100. In some embodiments k is an odd integerbetween 5 and 99. In some embodiments, each respective set of k-mersincludes at least eighty percent of the possible k-mers of length k ofthe corresponding sequence read 728 (1138). For example, referring toFIG. 10, if the sequence read has the sequence GATCCATCTT and k is setto 3, the corresponding set of k-mers includes at least 80 percent ofthe 8 possible k-mers, e.g., at least 6 of the 8 possible k-mers.

In some embodiments, each respective set of k-mers includes at leastsixty percent, at least seventy percent, at least eighty percent, atleast ninety percent, at least ninety-five percent, or all of thepossible k-mers of length k of the corresponding sequence read 728(1138). In some embodiments, a respective set of k-mers includes atleast sixty percent, at least seventy percent, at least eighty percent,at least ninety percent, at least ninety-five percent, or all of thepossible k-mers of length k of the first portion of the correspondingsequence read 728 (1138). In some embodiments, two or more sets ofk-mers includes at least sixty percent, at least seventy percent, atleast eighty percent, at least ninety percent, at least ninety-fivepercent, or all of the possible k-mers of length k of the correspondingsequence read 728 (1138).

Referring to FIG. 11C (1140), in some embodiments, the plurality ofsequence reads collectively provide at least 15× or at least 25×coverage for the larger contiguous nucleic acid 802, more than tenpercent or more than thirty percent of k-mers 746 in the plurality ofk-mers are from more than one sequence read 728 in the pluralitysequence reads, and the identifier 732 of each such sequence read 728for each k-mer 746 represented by more than one sequence read isretained.

In some embodiments, more than ten percent, more than twenty percent,more than thirty percent, more than forty percent, more than fiftypercent, more than sixty percent, more than seventy percent, or morethan eighty percent of the k-mers in the plurality of k-mers are frommore than one sequence read 728 in the plurality sequence reads. Ininstances where a respective k-mer 746 is found in more than onesequence read 728, the barcode (second portion) 732 of each sequenceread 728 is associated with the respective k-mer 746.

In some embodiments, the plurality of sequence reads collectivelyprovide at least 5×, 10×, 15×, 20×, 25×, or at least 30× coverage forthe larger contiguous nucleic acid 802.

Referring to FIG. 11C (1140), in some embodiments, the sequence reads728 in the plurality of sequence reads encode between 75 and 125 basesof the larger contiguous nucleic acid 802 and the value k is an oddinteger between 5 and 73 (1142). In some embodiments, the sequence reads728 in the plurality of sequence reads encode between 80 and 120 basesof the larger contiguous nucleic acid 802 and the value k is an oddinteger between 11 and 51. In some embodiments, the sequence reads 728in the plurality of sequence reads encode between 90 and 110 bases ofthe larger contiguous nucleic acid 802 and the value k is an odd integerbetween 13 and 45.

Referring to FIG. 11C (1144), in some embodiments, the respective set ofk-mers is created by hashing the first portion 730 each sequence read728 in the plurality of sequence reads according to a predeterminedk-mer length k thereby creating a respective set of k-mers for eachsequence read 728 in the plurality of sequence reads. One such method ofgenerating k-mers from a sequence read involves computing a hashfunction at each position of a sliding window of nucleotide length koverlayed on the first portion 730 of sequence read 728, in order toform a set of k-mers corresponding to the first portion 730 of thesequence read. In this method, each possible k-mer of length k is hashedusing a simple function, called the hash function. One possible hashfunction h(W) for nucleic acid sequences maps a k-mer of the form W=w₁w₁. . . w_(k) from the first portion 730 of the sequence read 728according to:

h(W)=ƒ(w _(k))+ƒ(w _(k−1))×4+ . . . +ƒ(w _(i))×4^(k−1)

where ƒ(W)=0, 1, 2, 3 for w_(i)=A, C, G, T. Other hash functions arepossible. In general any function that assigns a k-mer of the sequenceW=w₁w₁ . . . w_(k) from a sequence read to a unique number that can beused to reconstruct the actual nucleic acid sequence of the k-mer can beused. FIG. 9 shows the possible values of h(W) for the 64 possiblek-mers of length three. Accordingly, in some embodiments, the value h(W)for each k-mer 746 is stored in hash table 740 or some other form ofdata structure. In some embodiments, as illustrated in FIG. 7, for eachsequence read 728, the associated second portion 732 (e.g., bar code) ofthe partition from which the sequence read 728 is retained. Further, insome embodiments, the data structure for k-mers 740 further includes theposition of each k-mer 746 in the corresponding sequence read 728. Intypical embodiments, each possible k-mer in the first portion 730 ofsequence read 728 is converted to a k-mer 746. For instance, in anexample where the k-mer length is three and W=w₁w₁ . . . w_(k) (e.g.,the second portion 732 of the sequence read 728) is GATCCATCTT, thek-mers of FIG. 10 would be generated and hash values, e.g., h(W) values,for each of these k-mers would be generated.

Track the k-mers. Referring to FIG. 11D (1146), for each respectivek-mer 746 in the plurality of k-mers, an identity of each sequence read728 in the plurality of sequence reads that contains the respectivek-mer 746 and the identifier 732 of the set of sequence reads thatcontains the sequence read is tracked. For example, referring to FIG. 7,one way to accomplish this is to track all the k-mers 746 generated froma sequence read 728 along with the barcode 732. Alternative ways arepossible. For instance, a data structure, or collection of datastructures, that track all the sequence reads 728 that contain arespective k-mer 746, along with the barcodes 732 of these sequencereads 728, may be implemented. This data structure, or collection ofdata structures, would further track the sequence reads 728 that containall other respective k-mer 746, along with the barcodes 732 of thesesequence reads 728.

Graph the k-mers. Referring to FIG. 11D (1148), the plurality k-mers aregraphed as a graph comprising a plurality of nodes connected by aplurality of directed arcs. Each node comprises an uninterrupted set ofk-mers in the plurality of k-mers of length k with k−1 overlap. Each arcconnects an origin node to a destination node in the plurality of nodes.A final k-mer of an origin node has k−1 overlap with an initial k-mer ofa destination node. A first origin node has a first directed arc withboth a first destination node and a second destination node in theplurality of nodes (1148).

FIG. 14 illustrates one such graph 1400, which is known in the art as ade Bruijn graph. See Zerbino and Birney, “Velvet: Algorithms for de novoshort read assembly using de Bruijn graphs,” Genome Reach 2008,18:821-829, which is hereby incorporated by reference in its entirety.Each node 1402 represents a series of overlapping k-mers. In the case ofFIG. 14, k=5. Adjacent k-mers in a node 1402 overlap by k−1 nucleotides.The marginal information contained by a k-mer is its last (e.g., last3′) nucleotide. The sequence of those final nucleotides is called thesequence of the node 1402 b, or s(N). In FIG. 14, and in optionalembodiments, each node 1402 is attached to a twin node 1402 b, whichrepresents the reverse series of reverse complement k-mers. This ensuresthat overlaps between reads from opposite strands are taken intoaccount. Note that the sequences attached to a node and its twin do notneed to be reverse complements of each other. The union of a node 1402and its twin 1402 b is referred to a “block.” Nodes 1402 are connectedby a directed “arc” 1404. In that case, the last k-mer of an arc'sorigin node overlaps with the first of its destination node. Because ofthe symmetry of the blocks, if an arc goes from node 1402-1 to node1402-2, a symmetric arc goes from node 1402 b-2 to 1402 b-1. On thesenodes 1402 and arcs 1404, reads are mapped as “paths” traversing thegraph. Extracting the nucleotide sequence from a path is taken from theinitial k-mer of the first node and the sequences of all the nodes inthe path.

FIG. 14 illustrates a situation in which a first origin node has a firstdirected arc with both a first destination node and a second destinationnode in the plurality of nodes (1148). In particular node 1402-2 is anorigin node with a first directed arc 1404-2 to node 1402-3 and a seconddirected art 1404-3 to node 1402-4. This is because there is k−1 overlapbetween last k-mer of node 1402-2 (GATTG) and the first k-mer of node1402-3 (ATTGA), i.e., an overlap of ATTG, as well as an overlap betweenlast k-mer of node 1402-2 (GATTG) and the first k-mer of node 1402-4(ATTGC), i.e., an overlap of ATTG. Thus, there are two paths in graph1400, the path that comprises 1402-1→1404-1→1402-2→1404-2→1402-3 and thepath that comprises 1402-1→1404-1→1402-2→1404-3→1402-4. The disclosedmethods are useful in determining which path more likely represents thecorresponding sequence of the larger nucleic acid 802.

Determine which nodes to merge. Referring to FIG. 11D (1150), adetermination as to whether to merge the origin node with the firstdestination node or the second destination node is made in order toderive a contig sequence comprising (i) the origin node and (ii) one ofthe first destination node and the second destination node. The contigsequence that is more likely to be representative of the correspondingportion of the larger contiguous nucleic acid 802 is determined andselected. For example, referring to FIG. 14, a determination is made asto whether to merge node 1402-2 (origin node) and 1402-3 (firstdestination node) or to merge 1402-2 (origin node) and 1402-4 (seconddestination node). This determining 1150 advantageously uses at leastthe identifiers 732 of the sequence reads 728 for k-mers 740 in thefirst origin node, the first destination node, and the seconddestination node to identify the contig that is more likely to berepresentative of the corresponding portion of the larger contiguousnucleic acid 802.

In some embodiments, step 1150 is accomplished, at least in part, byevaluating the barcodes 732 associated with each k-mer in two or morepaths. For instance, in graph 1400, the barcodes 732 of the k-mers inpath 1402-1→1404-1→1402-2→1404-2→1402-3 and the barcodes 732 of thek-mers in path 1402-1→1404-1→1402-2→1404-3→1402-4 are evaluated. Sincethe source nucleic acid 802 typically includes multiple copies of thetarget nucleic acid (e.g., multiple copies of the same target chromosomeor other nucleic acid that is to be sequenced), there are typicallymultiple barcodes 732 for each k-mer in a node. Nevertheless, thecorrect path, that is the path that represents a true sequence found inthe larger contiguous nucleic acid 802, will have more barcodes incommon than paths that do not represent the true sequence found in thelarger contiguous nucleic acid 802. The actual method by which barcodesare used to identify the correct path is dependent upon a number ofvariables and so may vary.

In some embodiments, the more likely path, that is the better contigsequence comprising (i) the origin node and (ii) one of the firstdestination node and the second destination node, is selected byimposing a cut off, for instance, requiring at least 30 percent of thebarcodes representing any of the k-mers in the origin node and any ofthe k-mers in the selected destination node be common between the twonodes.

In some embodiments, the more likely path, that is the better contigsequence comprising (i) the origin node and (ii) one of the firstdestination node and the second destination node, is selected byimposing a cut off, for instance, requiring at least 10 percent of thebarcodes representing, at least 20 percent of the barcodes representing,at least 30 percent of the barcodes representing, at least 40 percent ofthe barcodes representing, at least 50 percent of the barcodesrepresenting or at least 60 percent of the barcodes representing any ofthe k-mers in the origin node and any of the k-mers in the selecteddestination node be common between the two nodes.

In some embodiments, the more likely path, that is the better contigsequence comprising (i) the origin node and (ii) one of the firstdestination node and the second destination node, is selected byidentifying the node pair that has the highest percentage of sharedbarcodes.

Referring to FIG. 11D (1152), in some embodiments, the first origin nodeand the first destination node are part of a first path in the graphthat includes one or more additional nodes other than the origin nodeand the first destination node. The first origin node and the seconddestination node are part of a second path in the graph that includesone or more additional nodes other than the origin node and the seconddestination node. This is illustrated in FIG. 14 as path1402-1→1404-1→1402-2→1404-2→1402-3 and path1402-1→1404-1→1402-2→1404-3→1402-4 where node 1402 is an additional nodein both instances. In some such embodiments, the determining of 1150comprises determining whether the first path is more likelyrepresentative of the larger contiguous nucleic acid than the secondpath by evaluating a number of identifiers 728 shared between the k-mersof the nodes of a first portion of the first path and the k-mers of thenodes of a second portion of the first path versus a number ofidentifiers shared between the k-mers of the nodes of a first portion ofthe second path and the k-mers of the nodes of a second portion of thesecond path (1152). In some embodiments, this is done on a relativebasis. For instance, of the total number of identifiers 746 associatedwith any of the k-mers in a path, what percentage of them are sharedbetween a first portion of the path and a second portion of the path.For example, referring to FIG. 15, consider an instance where path 1comprises nodes 1502, 1504, 1506, 1508, 1510, and path 2 comprises nodes1512, 1514, 1506, 1516, and 1518. Path 1 can be divided into a firstportion consisting of nodes 1502, 1504, and 1506 and a second portionconsisting of nodes 1508 and 1510. The identifiers 732 associated withany of the k-mers of nodes 1502, 1504, and 1506 are determined. Theidentifiers 732 associated with any of the k-mers of nodes 1508 and 1510are determined. This is repeated for the second path. That is, path 2 isdivided into a first portion consisting of nodes 1512, 1514, and 1506and a second portion consisting of nodes 1516 and 1518. The identifiers732 associated with any of the k-mers of nodes 1512, 1514, and 1506 aredetermined. The identifiers 732 associated with any of the k-mers ofnodes 1516 and 1518 are determined. The extent to which there is overlapbetween the identifiers 732 of the first and second portions of thefirst path is compared to the extent to which there is overlap betweenthe identifiers 732 of the first and second portions of the second path.For instance, consider the case in which thirty percent of the totalnumber of identifiers found in either the first or second portion of thefirst path is found in both the first and second portion of the firstpath. Further, forty-five percent of the total number of identifiersfound in either the first or second portion of the second path is foundin both the first and second portion of the second path. In thisinstance, the second path will be deemed to be more likelyrepresentative of a corresponding part of the larger contiguous nucleicacid 802. Other techniques for comparing the barcodes 732 of k-mers inpaths may be used, such as Bayesian analysis or Pearson's chi-squiredtest. The instance discovery is the use of the barcodes 732 to breakambiguity in the paths.

Referring to FIG. 11E (1154), in some embodiments, the first origin nodeand the first destination node are part of a first path in the graphthat includes one or more additional nodes other than the origin nodeand the first destination node. The first origin node and the seconddestination node are part of a second path in the graph that includesone or more additional nodes other than the origin node and the seconddestination node. The determining up-weights the first path relative tothe second path when the first path has higher average coverage than thesecond path (1154). Referring again to FIG. 15 to illustrate and againconsidering path 1 (1502, 1504, 1506, 1508, 1510), and path 2 (nodes1512, 1514, 1506, 1516, and 1518), if there is higher coverage by thek-mers of the nodes of path 1 relative to the k-mers of the nodes ofpath 2, this may be used as a tie breaker in instances where process1150 or 1152 of FIG. 11 was not able to identify a clear winner based onthe sequence identifiers alone.

Referring to FIG. 11E (1156), in some embodiments, the first origin nodeand the first destination node are part of a first path in the graphthat includes one or more additional nodes other than the origin nodeand the first destination node. The first origin node and the seconddestination node are part of a second path in the graph that includesone or more additional nodes other than the origin node and the seconddestination node. The determining up-weights the first path relative tothe second path when the first path represents a longer contiguousportion of the larger contiguous nucleic acid sequence than the secondpath (1156). In some embodiments, process 1156 is used as a tie breakerin instances where process 1150, 1152, or 1154 of FIG. 11 is not able toidentify a clear winner based on the sequence identifiers alone. In someembodiments, process 1156 is used in conjunction with processes 1150,1152, and/or 1154 of FIG. 11 to identify a winner using multiple sourcesof information.

Referring to FIG. 11E (1158), in some embodiments, a first k-mer 746 inthe first node is present in a sub-plurality of the plurality ofsequence reads 728 and the identity of each sequence read 728 in thesub-plurality of sequence reads is retained for the first k-mer 728 andused by the determining 1150 to determine whether the first path is morelikely representative of the larger contiguous nucleic acid sequencethan the second path (1158). This arises in many instances where thesample of the larger contiguous nucleic acid 802 that is partitionedinto fragments 804 in fact constitutes multiple copies of a targetnucleic acid (e.g., multiple copies of the same target chromosome orother nucleic acid that is to be sequenced). This results in multiplebarcodes 732 for each k-mer 746 in a node. As such, any of the processes1150, 1152, 1154, 1156 and/or 1158 are capable of taking this intoaccount.

Another aspect of the present disclosure provides a sequencing methodcomprising, at a computer system having one or more processors, andmemory storing one or more programs for execution by the one or moreprocessors, obtaining a plurality of sequence reads as disclosed abovein relation to 1106 of FIG. 11A. The plurality of sequence readscomprises a plurality of sets of sequence reads. Each respectivesequence read in a set of sequence reads includes (i) a unique firstportion that corresponds to a subset of a larger contiguous nucleic acidand (ii) a common second portion that forms an identifier that isindependent of the sequence of the larger contiguous nucleic acid andthat identifies a partition, in a plurality of partitions, in which therespective sequence read was formed. Each respective set of sequencereads in the plurality of sets of sequence reads is formed in apartition in the plurality of partitions. Each such partition includesone or more fragments of the larger contiguous nucleic acid that is usedas the template for each respective sequence read in the partition.

In the method, a respective set of k-mers is created for each sequenceread in the plurality of sequence reads as discussed above in relationto element 1138 of FIG. 11C. The sets of k-mers collectively comprise aplurality of k-mers. The identifiers 732 of the sequence read 728 foreach k-mer 746 in the plurality of k-mers is retained. The value k isless than the average length of the sequence reads in the plurality ofsequence reads. Each respective set of k-mers includes at least some of(e.g., at least eighty percent of) the possible k-mers of the firstportion of the corresponding sequence read.

In the method, there is tracked, for each respective k-mer in theplurality of k-mers, an identity of each of the sequence reads in theplurality of sequence reads that contains the respective k-mer. A firstpath is identified. The first path comprises a first set of k-mers withk−1 overlap in the plurality of k-mers (e.g., nodes 1502, 1504, 1506,1508, and 1510 of FIG. 15). A second path is identified. The second pathcomprises a second set of k-mers with k−1 overlap in the plurality ofk-mers (e.g., nodes 1512, 1514, 1506, 1516, and 1518 of FIG. 15). Thefirst path intersects the second path (e.g., as illustrated in FIG. 15),thereby forming the set of branch segments comprising: a left portion ofthe first path (nodes 1502 and 1504), a right portion of the first path(nodes 1508 and 1510), a left portion of the second path (nodes 1512 and154) and a right portion of the second path (1516 and 1518) where thechoice of what do with the bisecting node 1506 (e.g., not use it, put itin paths, or put it in one portion of each path) is applicationdependent. The identifiers associated with the k-mers of the set ofbranch segments are used to verify the connectivity of the branchsegments. In some embodiments, this comprises evaluating a number ofidentifiers shared between each possible branch segment pairs in the setof branch segments. In some instances, this may result in rerouting apath. For example, in evaluating the graph of FIG. 15, four possiblepaths are possible, as set forth in the following Table. As the tableindicates, path 1520 to 1522 (nodes 1502, 1504, 1506, 1508, and 1510)and path 1524 to 1526 (1512, 1514, 1506, 1516, and 1518 are verified ascorrect because they each have more overlapping barcodes, on a relativebasis, than path 1520 to 1526 and 1524 to 1522.

Number of Number of Number of Sequence Sequence Sequence Identifiers 732in Identifiers 732 in Identifiers 732 Left Path branch Left Path branchLeft Branch Right Branch that overlap left segment segment SegmentSegment and right branch 1520 1522 100 110 65 1520 1526 100 105 3 15241522 90 110 8 1524 1526 90 105 50

In some embodiments, additional information such as path lengths andpath coverage is used to verify these connections. It will beappreciated that any form of statistical analysis may be applied to theinformation such as that in the above table to verify the paths.

III. Exemplary Assembly Process

The following is an example of an overall de novo assembly process asdescribed above, is set forth below, and schematically illustrated inthe process flow chart shown in FIG. 4. As shown, following sequencingand read generation, the short read sequences are aggregated in a datastructure and subjected to pre-assembly processing at step 402. In thesereads, sequence data associated with functional components of thesequenced fragments, e.g., adapter sequences, read primer sequences,etc., are removed from the sequence reads. Duplicate reads may beeliminated by identifying those that are duplicated both in the firsttwo k-mers of the sense read and the partner or antisense read. Wherethe initial k-mers are common among duplicate reads and theircomplements, all but one can be removed as a duplicate using, e.g., AFastUniq algorithm, or other similar algorithms. In some cases,duplicate reads may get through the pre-assembly process, where one ormore of those duplicates include a sequencing error. Because these firstfew k-mers occur at the beginning of a sequencing run where errors arefewer, it would be expected that the probability of this occurring wouldbe low. In addition to duplicate reads, low complexity reads, e.g.,reads where a single base makes up 90% or more of the read, or readsthat are entirely or substantially made up of 2, 3, or 4 base repeatunits.

The reads are then subjected to an error correction algorithm at step404, as uncorrected errors in sequence reads result in fragmentedassemblies and add computational complexity. Different error correctionalgorithms are generally available (see, e.g., Tahir et al., “Review ofGenome Sequence Short Read Error Correction Algorithms”, Am. J.Bioinformatics Res. 2013; 3(1) 1-9, which is hereby incorporated hereinby reference) and include, for example the QUAKE, and standaloneALLPATHS-LG error correction algorithm.

Following the preassembly processing and error correction, using a k-mersize of, e.g., k=20 or k=24, a frequency spectrum of k-mers is generatedacross all of the aggregated reads at step 406. Assuming a typicalgenome profile, sequenced without significant bias, the expectationwould be for a Gaussian distribution of k-mer frequency, where thecenter of the peak indicates the mean k-mer coverage of the genome. Thesize of the k-mer used for calculation of the frequency spectrum maygenerally vary depending upon the complexity of the genome beinganalyzed, the expected level of sequencing coverage, and the like.

An initial assembly graph is then assembled at step 408 from the errorcorrected reads, using the k-mer assembly process described withreference to FIG. 1B. In some cases, longer k-mer sequences may be used,e.g., for sequence reads of 150 bases, k-mers of 40 or more, 50 or more,or 60 or more bases may be used. From the graph, unbranched paths arecollapsed into individual primary paths.

Hanging ends that represent relatively short branches, e.g., of 1, 2 or3 k-mers, are generally prevalent in the sequence and can result from anumber of origins, including from uncorrected reads. These short pathsare easily recognized and may be removed from the assembly graph, eitherbefore or after identifying the primary paths. In some cases, shortbranches may appear, but may be derived from missing sequence in theerror corrected reads, which can result from low coverage.

The barcode sequences coupled with the error corrected reads are thenassociated with their respective primary paths, at step 410 generating alist of the barcodes associated with each primary path. For each primarypath, a copy number of that primary path within the genome is estimated.As noted, the copy number of a given primary path will generally dependupon the complexity of the genome and the length and complexity of theprimary path. Copy number can generally be estimated from the number ofreads that touch a primary path, and comparing it to the mean k-mercoverage derived from the k-mer spectrum calculated at step 406. Shortprimary paths may generally be ignored, as determining their copy numberis more difficult since a much larger proportion of reads that touch onthose primary paths may only partially overlap them. From this, low copynumber primary paths, e.g., with a copy number approaching 1, alsoreferred to as CN1 paths, as noted above, may be identified at step 412.

The CN1 primary paths are then assembled in a scaffold with the aid ofthe associated barcode sequences. In particular, the CN1 primary paththat is associated with the largest number of barcodes is selected as aseed path at step 414. At step 416, the seed path is then extended toall other overlapping primary paths containing the common barcodesequence, considering each primary path only once, and ignoring primarypaths that have a barcode frequency below a desired level, e.g., lessthan half the barcode frequency in the seed path. FIG. 5 illustrates theprocess in greater detail. In particular, the first seed path isidentified at step 502. That first seed path is ten extended asdescribed for step 416, above. In particular, the extension is continuedthrough primary paths and additional CN1 paths at step 504, until theend of a given branch is reached. The path is then trimmed back to thelast CN1 path through which the path traveled at step 506, such that theassembled sequence will always begin an end on a CN1 path, and beanchored around the seed path.

The paths through the CN1 paths are then condensed by removing all ofthe non-CN1 paths, then merging together to form a single scaffold thatdefines the order to the CN1 paths around the first seed path at step508. These CN1 paths are then designated as used.

The process is then repeated at step 512 using the CN1 path, from theremaining CN1 paths, that represents the highest remaining barcodefrequency as the next seed path, and scaffolding against the remainingsets of CN1 paths. This is continued until all CN1 paths are designatedas used. The set of condensed primary paths is then further merged toprovide a final path or scaffold that gives the relative order of all ofthe CN1 paths in the genome at step 514. This forms the anchor scaffoldwhich provides the basis for final assembly.

Once the CN1 path scaffold is assembled, local assembly is carried outto fill in the scaffold gaps between CN1 paths at step 420. For each gapin the scaffold, the adjacent primary paths are selected and thebarcodes associated with them are identified. The group of reads thatincludes these barcodes are then used in conjunction with the adjacentCN1 paths to assemble a local primary path assembly graph in the samemanner as used to assemble the global primary path graph. From each CN1path, all paths that connect to the other primary path are identified.In many cases, only a single path will be identified allowing the use ofthat path as the patch to the gap. Where more than one path isidentified between two adjacent CN1 primary paths, the path with thestrongest read support is used to patch together the adjacent CN1primary paths and close the gap. In cases where read support does notallow selection as between two paths, both paths may be included in thegraph, or one can allow the gap to remain open.

From the full assembly, a linear graph of the entire genome is createdthat, ideally, includes no remaining gaps or doubled paths. However, inmany cases, the linear graph may include some locations of dual paths orremaining gaps, but the overall order of the sequence is known. Forremaining gaps or double paths, one may further examine uncorrectedsequence data, or discarded sequence data to determine if any gaps ordoubles may be informed by those sequences that were removed from theprocess early on. Alternatively, other disambiguation techniques may beused, such as using copy number information to inform ambiguousassemblies, or using read pair information to evaluate relativelikelihood of multiple solutions for ambiguous assemblies.

In certain aspects, the methods provided herein are computer-implementedmethods, wherein at least one or more steps of the method are carriedout by a computer program. In some embodiments, the methods providedherein are implemented in a computer program stored on computer-readablemedia, such as the hard drive of a standard computer. For example, acomputer program for determining at least one consensus sequence fromreplicate sequence reads can include one or more of the following: codefor providing or receiving the sequence reads, code for identifyingregions of sequence overlap between the sequence reads, code foraligning the sequence reads to generate a layout, contig, or scaffold,code for consensus sequence determination, code for converting ordisplaying the assembly on a computer monitor, code for applying variousalgorithms described herein, and a computer-readable storage mediumcomprising the codes.

In some embodiments, a system (e.g., a data processing system) thatdetermines at least one assembly from a set of replicate sequencesincludes a processor, a computer-readable medium operatively coupled tothe processor for storing memory, wherein the memory has instructionsfor execution by the processor, the instructions including one or moreof the following: instructions for receiving input of sequence reads,instructions for overlap detection between the sequence reads,instructions that align the sequence reads to generate a layout, contig,or scaffold, instructions that apply a consensus sequence algorithm togenerate at least one consensus sequence (e.g., a “best” consensussequence, and optionally one or more additional consensus sequences),instructions that compute/store information related to various steps ofthe method, and instructions that record the results of the method.

In certain embodiments, various steps of the processes described hereinutilize information and/or programs and generate results that are storedon computer-readable media (e.g., hard drive, auxiliary memory, externalmemory, server, database, portable memory device and the like. Forexample, information used for and results generated by the methods thatcan be stored on non-transitory computer-readable media include but arenot limited to input sequence read information, set of pair-wiseoverlaps, newly generated consensus sequences, quality information,technology information, and homologous or reference sequenceinformation.

In some aspects, the invention includes an article of manufacture fordetermining at least one sequence assembly that includes amachine-readable medium containing one or more programs which whenexecuted implement the steps of the invention as described herein.

As will be understood to practitioners in the art from the teachingsprovided herein, the invention can be implemented in hardware and/orsoftware. In some embodiments of the invention, different aspects of theinvention can be implemented in either client-side logic or server-sidelogic. As will be understood in the art, the invention or componentsthereof may be embodied in a fixed media program component containinglogic instructions and/or data that when loaded into an appropriatelyconfigured computing device cause that device to perform according tothe invention. As will be understood in the art, a fixed mediacontaining logic instructions may be delivered to a viewer on a fixedmedia for physically loading into a viewer's computer or a fixed mediacontaining logic instructions may reside on a remote server that aviewer accesses through a communication medium in order to download aprogram component, or to which the user uploads data to be processedusing the above described hardware and/or software, at the remotelocation or locations, in accordance with the processes set forthherein.

Information appliances (or digital devices) that are generally used inconjunction with the processes describe herein include conventionallogic systems, such as conventional computer systems, including bothindividual or personal computers, networked computers, computermainframes, and the like. Interaction with these processes may not belimited to such conventional computing systems, but may additionally oralternatively include any information appliance for interacting with aremote data application, and could include such devices as a digitallyenabled television, cell phone/smart phone, personal digital assistant,tablet, wearable electronic devices, e.g., Google Glass, smart watch,etc. Programming for carrying out the processes described herein on anyof the foregoing types of devices may reside within the memory of thedevice itself (either main memory or auxiliary memory), or it may beaccessed from other locations and devices, e.g., computers through,e.g., an API on the device, and an appropriate communications network,e.g., LAN, WAN, Wi-Fi, cellular, Bluetooth or other communicationssystem.

The E. coli genome (strain K12_DH10B) was used to model the de-novoassembly processes described herein. Sixty physical copies of the genomewere sheared to an average size of 50 kilobases (standard deviation 5kilobases) and partitioned at random into 5000 partitions, eachcontaining a unique barcode. The process of the creation of abarcode-sequencing library was simulated within each partition. This wasfollowed by simulation of sequencing of this library utilizing 150 bppaired-end reads to a mean depth of 30×. The simulation of thesequencing process incorporated an empirical error rate model derivedfrom real sequencing data. FIG. 6 shows a de novo assembly plot for theabove-described modeled data. As shown, the assembly resulted in N50contigs of 750 kb as compared to N50 contigs of 60 kb for data basedupon short read sequence alone, e.g., without barcodes.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. For example, particle delivery canbe practiced with array well sizing methods as described. Allpublications, patents, patent applications, and/or other documents citedin this application are incorporated by reference in their entirety forall purposes to the same extent as if each individual publication,patent, patent application, and/or other document were individually andseparately indicated to be incorporated by reference for all purposes.

1. A sequencing method of assembling nucleic acid sequence readscomprising: at a computer system having one or more processors, andmemory storing one or more programs for execution by the one or moreprocessors: obtaining a plurality of sequence reads derived from alarger contiguous nucleic acid, wherein two or more sequence readsderived from a common fragment of the larger contiguous nucleic acidcomprise a common barcode sequence, identifying a first subset ofsequence reads in the plurality of sequence reads that comprise bothoverlapping sequences and a common barcode sequence; and aligning thefirst subset of sequence reads to provide a contiguous linear nucleicacid sequence.
 2. The method of claim 1, further comprising repeatingthe identifying and aligning steps with a plurality of different subsetsof sequence reads to provide a plurality of contiguous linear nucleicacid sequences.
 3. The method of claim 2, further comprising orderingthe plurality of different contiguous linear nucleic acid sequences in asequence context within the larger contiguous nucleic acid.
 4. Themethod of claim 3, wherein the ordering comprises mapping the pluralityof different contiguous linear nucleic acid sequences against areference sequence.
 5. The method of claim 3, wherein the orderingcomprises: identifying one or more sequence reads that comprise abarcode sequence common to a first contiguous linear nucleic acidsequence, but include overlapping sequences with a second contiguouslinear nucleic acid sequence; and identifying the first and secondcontiguous linear nucleic acid as structurally linked.
 6. A method ofassembling nucleic acid sequence reads into larger contiguous sequences,comprising: at a computer system having one or more processors, andmemory storing one or more programs for execution by the one or moreprocessors: obtaining a plurality of sequence reads derived from alarger contiguous nucleic acid, identifying a first subsequence from aset of overlapping sequence reads in the plurality of sequence reads;extending the first subsequence to one or more adjacent or overlappingsequences based upon the presence of a barcode sequence on the adjacentsequence that is common to the first subsequence; and providing a linearnucleic acid sequence that comprises the first subsequence and the oneor more adjacent sequences.
 7. A sequencing method comprising, at acomputer system having one or more processors, and memory storing one ormore programs for execution by the one or more processors: (A) obtaininga plurality of sequence reads, wherein the plurality of sequence readscomprises a plurality of sets of sequence reads, each respectivesequence read in a set of sequence reads includes (i) a first portionthat corresponds to a subset of a larger contiguous nucleic acid and(ii) a common second portion that forms an identifier that isindependent of the sequence of the larger contiguous nucleic acid andthat identifies a partition, in a plurality of partitions, in which therespective sequence read was formed, and each respective set of sequencereads in the plurality of sets of sequence reads is formed in apartition in the plurality of partitions and each partition includes oneor more fragments of the larger contiguous nucleic acid that is used asthe template for each respective sequence read in the partition; (B)creating a respective set of k-mers for each sequence read in theplurality of sequence reads, wherein the sets of k-mers collectivelycomprise a plurality of k-mers, the identifiers of the sequence readsfor each k-mer in the plurality of k-mers is retained, k is less thanthe average length of the sequence reads in the plurality of sequencereads, and each respective set of k-mers includes at least eightypercent of the possible k-mers of length k of the first portion of thecorresponding sequence read; (C) tracking, for each respective k-mer inthe plurality of k-mers, an identity of each sequence read in theplurality of sequence reads that contains the respective k-mer and theidentifier of the set of sequence reads that contains the sequence read;(D) graphing the plurality k-mers as a graph comprising a plurality ofnodes connected by a plurality of directed arcs, wherein each nodecomprises an uninterrupted set of k-mers in the plurality of k-mers oflength k with k−1 overlap, each arc connects an origin node to adestination node in the plurality of nodes, a final k-mer of an originnode has k−1 overlap with an initial k-mer of a destination node, and afirst origin node has a first directed arc with both a first destinationnode and a second destination node in the plurality of nodes; and (E)determining whether to merge the origin node with the first destinationnode or the second destination node in order to derive a contig sequencethat is more likely to be representative of a portion of the largercontiguous nucleic acid, wherein the contig sequence comprises (i) theorigin node and (ii) one of the first destination node and the seconddestination node, wherein the determining uses at least the identifiersof the sequence reads for k-mers in the first origin node, the firstdestination node, and the second destination node.
 8. The sequencingmethod of claim 7, wherein, the first origin node and the firstdestination node are part of a first path in the graph that includes oneor more additional nodes other than the origin node and the firstdestination node, the first origin node and the second destination nodeare part of a second path in the graph that includes one or moreadditional nodes other than the origin node and the second destinationnode, and the determining (E) comprises determining whether the firstpath is more likely representative of the larger contiguous nucleic acidthan the second path by evaluating a number of identifiers sharedbetween the k-mers of the nodes of a first portion of the first path andthe k-mers of the nodes of a second portion of the first path versus anumber of identifiers shared between the k-mers of the nodes of a firstportion of the second path and the k-mers of the nodes of a secondportion of the second path.
 9. The sequencing method of claim 7, whereinthe first origin node and the first destination node are part of a firstpath in the graph that includes one or more additional nodes other thanthe origin node and the first destination node, the first origin nodeand the second destination node are part of a second path in the graphthat includes one or more additional nodes other than the origin nodeand the second destination node, and the determining (E) up-weights thefirst path relative to the second path when the first path has higheraverage coverage than the second path.
 10. The sequencing method ofclaim 7, wherein the the first origin node and the first destinationnode are part of a first path in the graph that includes one or moreadditional nodes other than the origin node and the first destinationnode, the first origin node and the second destination node are part ofa second path in the graph that includes one or more additional nodesother than the origin node and the second destination node, and thedetermining (E) up-weights the first path relative to the second pathwhen the first path represents a longer contiguous portion of the largercontiguous nucleic acid sequence than the second path.
 11. Thesequencing method of claim 7, wherein a first k-mer in the first node ispresent in a sub-plurality of the plurality of sequence reads and theidentity of each sequence read in the sub-plurality of sequence reads isretained for the first k-mer and used by the determining (E) todetermine whether the first path is more likely representative of thelarger contiguous nucleic acid sequence than the second path.
 12. Thesequencing method of claim 7, wherein a partition in the plurality ofpartitions comprises at least 1000 molecules with the common secondportion, and each molecule in the at least 1000 molecules furthercomprises a primer sequence complementary to at least a portion of thelarger contiguous nucleic acid.
 13. The sequencing method of claim 7,wherein a partition in the plurality of partitions comprises at least1000 molecules with the common second portion, and each molecule in theat least 1000 molecules further comprises a primer site and asemi-random N-mer priming sequence that is complementary to part of thelarger contiguous nucleic acid.
 14. The sequencing method of claim 7,wherein the one or more fragments of the larger contiguous nucleic acidin a partition in the plurality of partitions are greater than 50kilobases in length.
 15. The sequencing method of claim 7, wherein theone or more fragments of the larger contiguous nucleic acid in apartition in the plurality of partitions are between 20 kilobases and200 kilobases in length.
 16. The sequencing method of claim 7, whereinthe one or more fragments of the larger contiguous nucleic acid in apartition in the plurality of partitions consists of between 1 and 500different fragments of the larger contiguous nucleic acid.
 17. Thesequencing method of claim 7, wherein the one or more fragments of thelarger contiguous nucleic acid in a partition in the plurality ofpartitions consists of between 5 and 100 fragments of the largercontiguous nucleic acid.
 18. The sequencing method of claim 7, whereinthe plurality of sequence reads is obtained from less than 5 nanogramsof nucleic or ribonucleic acid.
 19. The sequencing method of claim 7,wherein the identifier in the second portion of each respective sequenceread in the set of sequence reads encodes a common value selected fromthe set {1, . . . , 1024}, the set {1, . . . , 4096}, the set {1, . . ., 16384}, the set {1, . . . , 65536}, the set {1, . . . , 262144}, theset {1, . . . , 1048576}, the set {1, . . . , 4194304}, the set {1, . .. , 16777216}, the set {1, . . . , 67108864}, or the set {1, . . . ,1×10¹²}.
 20. The sequencing method of claim 7, wherein the identifier isan N-mer, and N is an integer selected from the set {4, . . . , 20}.21.-44. (canceled)